Enzymatic Synthesis Of Nucleic Acids Research Articles

Alternative solution of the simplest model of enzymatic synthesis of nucleic acids by homotopy perturbation method.

Development of methods for obtaining approximate analytical solutions of nonlinear differential equations and their systems is a rapidly developing field of mathematical physics. Earlier, an approximate solution of the simplest system of kinetic enzymatic equations for calculating dynamics of complementary strands of nucleic acids was obtained. In this study, we consider an alternative approach to selecting the basic linear approximation of the used method, which makes it possible to obtain more accurate analytical solutions of the set problem.

Approximate analytic solution of a simple system of kinetic equations describing the enzymatic synthesis of nucleic acids.

Macroscopic kinetic models describing the process of polymerase chain reaction (PCR) are currently solved only by numerical methods, which hampers the development of effective software algorithms for processing the results of the reaction. This paper considers the application of the homotopy perturbation method for obtaining approximate analytical solution of the simplest system of enzymatic kinetic equations describing the synthesis of nucleic acid molecules during PCR. The resulting approximate analytic solution with high accuracy reproduces the results of a numerical solution of the system in a wide range of ratios of enzyme and substrate concentrations both for the case of a large excess of the substrate over the enzyme and vice versa.

ChemInform Abstract: Microwave‐Assisted Rapid Enzymatic Synthesis of Nucleic Acids.

AbstractThe microwave‐induced enhancement of reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus‐reverse transcriptase is reported.

Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids

ABSTRACTHerein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10–50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

ChemInform Abstract: Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages.

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for structural mutagenesis by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

Information‐bearing nucleic acids display universal 3′‐5′ linkages, but regioisomeric 2′‐5′ linkages occur sporadically in non‐enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site‐specific 2′‐5′ linkages by an engineered polymerase using 3′‐deoxy‐ or 3′‐O‐methyl‐NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′‐5′ linked DNA with good fidelity. This enables a fast and simple method for “structural mutagenesis” by the position‐selective incorporation of 2′‐5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild‐type base sequence as we demonstrate for the 10–23 RNA endonuclease DNAzyme.

Polymerase-Dependent DNA Synthesis from Phosphoramidate-Activated Nucleotides

Nucleoside triphosphate mimetics, which are substrates for polymerases, can be used in the enzymatic synthesis of nucleic acids. Alternatively, they might also become reversible or irreversible enzyme inhibitors. In order to analyze the effects of 5′-phosphoramidate modification of deoxynucleotide in DNA synthesis, 3-phosphono-L-Ala-dNMP (N = A, T, or G) were evaluated as substrates of HIV-1 RT, Vent (exo−), and Therminator polymerase, respectively. The DNA-dependent DNA polymerase activity is significantly higher for Vent exo− polymerase than for HIV-1 RT, which is reflected by the capacity of Vent exo− polymerase to efficiently synthesize DNA without stalling effects. In addition, Vent (exo−) polymerase proved to be more accurate than Therminator polymerase, based on Watson–Crick base-pairing. The optimal yield (88%–97%) of full-length elongation can be obtained in 60 minutes by Vent (exo−) polymerase at 0.025 U/μL, with the phosphoramidate analogues as substrates. These data led us to conclude that the optimal pyrophosphate mimetic for the enzyme-catalyzed synthesis of DNA is polymerase dependent.

3-Phosphono-l-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase

A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of deoxyadenosine triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 μM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson-Crick rules.

METABOLISM OF NUCLEIC ACIDS.

Enzymatic synthesis of nucleic acids. Template function of primers.-With few exceptions, observations on DNA-synthesizing enzymes support base complementarity (1 , 2) as the code for the template function of their primers. Extension of this coding principle to DNA primers of DNA-dependent RNA polymerases provides strong evidence for the concept of messenger RNAs (3) . These concepts continue to be fruitful, but certain findings require modification or at least flexibility in the formulation of the original postu­ lates. Largely unknown is the detailed mechanism of the interactions which result in the final correlation between the structures of the template and those of the product, i .e . , in the respective code. In the absence of evidence for the intermediary formation of covalently linked compounds between template and precursors, various physical ap­ proaches and criteria are being explored, as base complementarity has been observed in complexes formed nonenzymatically between simple purine and pyrimidine derivatives (4, 4a) as well as between polynucleotide chains of high molecular weights. The specificity of the nonenzymatic interaction be­ tween two complementary polynucleotide strands (or perhaps between com­ plementary portions of two strands) is illustrated by the nonenzymatic for­ mation of at least three types of complexes between respective comple­ mentary polynucleotide chains: (a) renaturation of heat-denatured DNA double strands, (b) hybrid formation between single-stranded deoxyribo­ polynucleotide chains containing complementary purines and pyrimidines other than the four principal DNA-constituent bases (e.g . , hybrid formation between the strictly alternating copolymer of deoxyadenylic and deoxy­ thymidylic acids and the strictly alternating copolymer of deoxyadenylic and deoxy-5-bromouridylic acids), (c) hybrid formation between complementary deoxyriboand ribopolynuc1eotide chains. This type of hybrid was discovered by Rich (5), who demonstrated inter­ action between r-poly A and synthetic deoxythymidylate oligonucleotides on the basis of hypochromicity and sedimentation behavior. Schildkraut et al . (6) obtained hybrids between r-poly C and d-poly G by annealing. They ob­ served that the resistance of complementary complexes between riboand deoxyribopolynucleotide chains against ribonuclease is an important cri­ terion for hybrid formation. Presumably owing to the requirement of com­ plementarity, certain natural RNA specimens hybridize only with homol­ ogous, but not with heterologous, DNA. On this basis, Spiegelman and his

Enzymatic synthesis of nucleic acids

personal int erest and also avoids overlap with the review Metabolism oj Nucleic Acids in this volume (1). Additional discussion of nucleic acid biosynthesis can be found in several recent reviews (2, 3) (see also reviews in vol. III of The Nucleic Acids, Academic Press Inc., New York, 1960), The last few years have produced not able advances in our understanding of the pathways of nucleic acid biosynthesis. Although an enzyme for ribo­ polynucleotide syn thesis was discovered first, knowle dge of the mechanism of RNA biosynthes is is much less complete than it is for DNA. DNA con­ tained within the chromosomes can be regarded as a single bioche mical and molecular unit, whereas cellular RNA is physically and metabo lically heterogeneous. It may well be, therefore, that in contrast to DNA synthe sis. which app ears to proceed by a unique mechanism, several different enzyme systems are involved in RNA synthe sis.

Enzymatic synthesis of nucleic acids.

personal int erest and also avoids overlap with the review Metabolism oj Nucleic Acids in this volume (1). Additional discussion of nucleic acid biosynthesis can be found in several recent reviews (2, 3) (see also reviews in vol. III of The Nucleic Acids, Academic Press Inc., New York, 1960), The last few years have produced not able advances in our understanding of the pathways of nucleic acid biosynthesis. Although an enzyme for ribo­ polynucleotide syn thesis was discovered first, knowle dge of the mechanism of RNA biosynthes is is much less complete than it is for DNA. DNA con­ tained within the chromosomes can be regarded as a single bioche mical and molecular unit, whereas cellular RNA is physically and metabo lically heterogeneous. It may well be, therefore, that in contrast to DNA synthe sis. which app ears to proceed by a unique mechanism, several different enzyme systems are involved in RNA synthe sis.