Polynucleotides synthesis

K. Tajima and T. Hata. Simple Protecting Group Protection-Purification Handle for Polynucleotide Synthesis, II. Bull. Chem. Soc. Jpn., 45, 2608 (1972). 

Shuman, S. (1994). Novel approach to molecular cloning and polynucleotide synthesis using vaccinai DNA topoisomerase. J. Biol. Chem. 269, 32678-32684. 

The first enzyme discovered that could catalyze polynucleotide synthesis was a bacterial enzyme called polynucleotide phosphorylase. This enzyme, isolated by Severo Ochoa and Marianne Grunberg-Manago in 1955, could make long chains of 5 -3 -linked polyribonucleotides starting from nucleoside diphosphates. However, there was no template requirement for this synthesis, and the sequence was uncontrollable except in a crude way by adjusting the relative concentrations of different nucleotides in the starting materials. 

Khorana, H. G. Polynucleotide synthesis and the genetic code. Federation Proc. 24, 1473 (1965). 

CTP), which are the building blocks for polynucleotide synthesis. The rate of RNA synthesis (lower part) is measured through incorporation of radioactive GTP into the newly produced RNA molecules. The rate of replication shows stepwise increase. An early decrease is observed, because first a quasi-species is formed by the master sequence through production of mutants of lower fitness. The figure is redrawn from the data in [12]. 

Since the early work of Elmore and Todd,23 several eminent groups have made important, independent contributions to this development,11 24-26 but a great many advances have relied heavily on Khorana s methodology, even when they did not actually emanate from his laboratories. The parallelism between the development of polynucleotide synthesis and that of polypeptide synthesis is obvious, and many of the early approaches were derived from consideration of analogous types of reaction in the peptide field. It remains to be seen whether advances in the techniques of polynucleotide synthesis will ever make this type of compound more accessible than synthetic poly-... 

The same principles found an even more spectacular application in the area of polynucleotide synthesis. Most importantly, it was recognized that the use of various types of solid support offered a number of promising strategic opportunities in other areas of organic synthesis. Thus the successful solution of what appeared to be initially a purely technical problem, of limited significance, led ultimately to results of enormous scientific importance. 

Arylsulphonyl 1,2,4-triazolides (35), prepared from the appropriate aryl-sulphonyl chloride and IjH-1,2,4-triazole, can be used to activate phospho-monoesters or -diesters for condensation reactions in polynucleotide synthesis. Yields for condensations involving guanosine residues are better than those obtained using aryl sulphonyl chlorides. 

Polynucleotide synthesis. Narang and collaborators have reported the synthesis of a pentadecanucleotide of thymidine by a modified phosphotriester method. 

Miyoshi KI, Huang TH, Itakura K. Solid-phase synthesis of polynucleotides Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester approach. Nucleic Acids Res 8 5491-5505, 1980. 

Michalski and coworkers have shown that sulphuryl chloride fluoride (25) is a convenient reagent for preparing both simple phosphorofluoridates and more complex carbo-hydrate-substituted phosphorofluoridates for use in polynucleotide synthesis. Using phosphorus (III) trimethylsilyl esters (equation 20), products of very high purity are obtained in excellent yields under mild reaction conditions. Alternatively, phosphorus(V) thionophosphate or selenophosphate esters (equation 21) and 25 also give the corresponding phosphorofluoridates in high yields. ... 

As does all polynucleotide synthesis, the reaction proceeds in the 5 to 3 direction. 

Equilibrium for the reaction catalyzed by polynucleotide phosphorylase lies toward the direction of RNA degradation rather than synthesis. High concentrations of ribonucleo-side diphosphates are required to achieve the net synthesis of RNA and it is likely that their concentrations in the cell are not sufficient to drive net polynucleotide synthesis. Also, polynucleotide phosphorylase does not use a template, so the polyribonucleotides it synthesizes contain random sequences, which makes them of no value for protein synthesis. The cell uses polynucleotide phosphorylase as a degradative enzyme in conjunction with other nucleases that regulate the lifetimes of RNA molecules, including mRNA. In bacteria mRNA lifetimes are relatively short. 

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