Enzymatic Polynucleotide Syntheses

Three types of enzymatic polynucleotide syntheses can be distinguished de novo syntheses, primer-dependent syntheses, and template-dependent syntheses. Templates are polynucleotides whose nucleotide sequences are being copied and which are not incorporated by covalent bonds into the polynucleotide being produced. If, however, the added polynucleotide is... 

Nucleic acids are the molecules of the genetic apparatus. They direct protein biosynthesis in the body and are the raw materials of genetic technology (see Genetic engineering). Most often polynucleotides are synthesized microbiologicaHy, or at least enzymatically, but chemical synthesis is possible. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Langer, P.R., Waldrop, A.A., and Ward, D.C. (1981) Enzymatic synthesis of biotin-labeled polynucleotides Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633-6637. 

Figure 10.3 Enzymatic synthesis of poly(adenylic acid) in self-reproducing oleate liposomes (redrawn from Walde et al., 1994a). (a) The ADP penetrates (sluggishly) the liposome bilayer, (b) in the presence of polynucleotide phosphorylase, ADP is converted in poly(A), which remains entrapped in the liposome, (c) Polycondensation of ADP goes on simultaneously with the self-reproduction of liposomes (A is the membrane precursor, oleic anhydride, which, once added, induces the self-reproduction of liposomes S, surfactant, in this case oleate, which is the hydrolysis product of A on the bilayer E is polynucleotide phosphorylase).

Another important technique was based on the observation that synthetic trinucleotides induced the binding to ribosomes of tRNA molecules that were "charged" with their specific amino acids 38/39 For example, the trinucleotides UpUpU and ApApA stimulated the binding to ribosomes of 14C-labeled phenylalanyl-tRNA and lysyl-tRNA, respectively. The corresponding dinucleotides had no effect, an observation that not only verified the two codons but also provided direct evidence for the triplet nature of the genetic code. Another powerful approach was the use of artificial RNA polymers, synthesized by combined chemical and enzymatic approaches.40 For example, the polynucleotide CUCUCUCUCU led to the synthesis by ribosomes of a regular alternating polypeptide of leucine and serine. 

Growing membrane systems have been used to obtain artificial infrabiological systems. Walde et al. [47] have carried out the synthesis of polyadenylic acid in self-reproducing vesicles [48], in which the enzyme polynucleotide phosphorylase carried out the synthesis of poly-A, and membrane vesicle multiplication was due to the hydrolysis of externally provided oleic anhydride to oleic acid. The snag is that the enzyme component is not auto-catalytic. Enzymatic RNA replication in vesicles [49] suffers from the same problem. It is also not known whether redistribution of the entrapped enzymes into newly formed vesicles occurs or not. An affirmative answer would be evidence for vesicle reproduction by fission. 

Different types of antitemplates can be and, presumably, will be, synthesized by employing other chemical reactions that are sufficiently mild and selective for the modification of polynucleotides. Another approach to this problem is, of course, to synthesize an appropriately modified nucleoside triphosphate and utilize it as one of the substrates in a DNA or RNA polymerizing system, in the presence of the specific template for which an antitemplate is to be made. The enzymatic synthesis method has some obvious advantages however, the inhibitory activity of the product (antitemplate) may limit its applications. 

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. 

When chemical and enzymatic methods for NTP synthesis are compared[941, enzymatic techniques provide the most convenient route to CTP and GTP, whereas chemical deamination of CTP is the best method for preparing UTP 94L ATP is relatively inexpensive from commercial sources, although it has been synthesized enzymatically from AMP on 50 mmol scale. Mixtures of NTPs can be prepared from RNA by sequential nuclease Pi, polynucleotide phosphorylase, and pyruvate kinase-catalyzed reactions11101. This mixture can be selectively converted to a sugar nucleotide using a particular sugar nucleoside diphosphate pyrophosphorylase11101. 

Increased efficiency by the enzymatic synthesis of amino acids and peptides, novel carbohydrates and oligosaccharides, nucleotides and polynucleotides, glycolipids and peptidoglycans. 

Polynucleotides possess a great many reactive groups (see Figure 29-1), which must be protected before a conventional chemical synthesis of polynucleotides via the sugar-phosphate polycondensation reaction can be performed. Consequently, an enzymatic synthesis is generally preferred. 

Kornberg, A. Pathways of enzymatic synthesis of nucleotides and polynucleotides. In The chemical basis of heredity (McElroy, W.D., and Glass, B., eds.), Symp., p. 579-608. Baltimore The Johns Hopkins University Press 1957... 

Enzymatic Synthesis. DNA and RNA are biosynthesized by polymerase enzymes. RNA is synthesized from a DNA strand with the assistance of an RNA polymerase enzyme. Inversely, DNA can be synthesized from an RNA strand with a reverse transcriptase (RT) enzyme. This mechanism is important because of retroviruses (such as HIV) in which RT makes up part of the viral replication cycle. (HIV-RT is an important target for anti-HIV therapy.) In any enzymatic synthesis of DNA or RNA, it is critical to the life of the organism that these enzymes synthesize their respective polynucleotides in a specific sequence of monomer units. The enzymes are able to accomplish this by utilizing a template strand to direct the synthesis. By reading the sequence of nucleobases on the template, the enzymes select the complementary monomer for incorporation into the propagating strand (Fig. 9). It was widely accepted that the polymerase enzymes accomplish this by matching the nucleotide to the template on the basis of complementary... 

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