scholarly journals Study of the multiple incorporation of modified nucleotides into the growing DNA strand

2021 ◽  
Vol 16 (2) ◽  
pp. 148-155
Author(s):  
O. S. Volkova ◽  
A. V. Chudinov ◽  
S. A. Lapa
Keyword(s):  
Primer Extension ◽  
Enzymatic Reactions ◽  
Synthetic Dna ◽  
Modified Dna ◽  
Substrate Properties ◽  
Enrichment Technology ◽  
Polymerase Chain ◽  
Dna Strand ◽  
Exponential Enrichment ◽  
Derivatives Of

Objectives. This study investigated the substrate properties of the modified derivatives of triphosphates of purine and pyrimidine deoxynucleosides (5-propynyl-2’-deoxyuridine-5’-triphosphate, 5-propynyl2’-deoxycytidine-5’-triphosphate, 5-methyl-2’-deoxycytidine-5’-triphosphate, and N6-methyl-2’-deoxyadenosine-5’-triphosphate) during their simultaneous incorporation in enzymatic reactions (polymerase chain and primer extension reactions).Methods. The real-time polymerase chain and primer extension reactions were used to study the substrate efficiency of modified deoxynucleotide triphosphates. Various pairwise combinations of modified derivatives were used; specially designed synthetic DNA fragments and libraries for the Systematic Evolution of Ligands by Exponential Enrichment technology were used as templates. Reactions were conducted using DNA polymerases: Taq, Vent (exo-), DeepVent (exo-), and KOD XL.Results. In each case, a pair of compounds (modified dUTP + dCTP, dUTP + dATP, and dCTP + dATP) was selected to study the simultaneous incorporation into the growing DNA strand. The most effective combinations of nucleotides for simultaneous insertion were dU and dC, having 5-propynyl substitution. The Vent (exo-) DNA polymerase was found as the most effective for the modified substrates.Conclusions. The selected compounds can be used for the enzymatic preparation of modified DNA, including aptamers with extended physicochemical properties. 

scholarly journals Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

2018 ◽  
Vol 115 (40) ◽  
pp. 9992-9997 ◽  
Author(s):  
Heike Maria Kropp ◽  
Simon Leonard Dürr ◽  
Christine Peter ◽  
Kay Diederichs ◽  
Andreas Marx
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Ternary Complexes ◽  
Structural Basis ◽  
Modified Nucleotides ◽  
Molecular Mechanics Calculations ◽  
Modified Dna ◽  
Substrate Properties ◽  
Dna Strand ◽  
Dna Substrate

DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.

scholarly journals Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

2016 ◽  
Vol 12 ◽  
pp. 2588-2601 ◽  
Author(s):  
Vladimir A Stepchenko ◽  
Anatoly I Miroshnikov ◽  
Frank Seela ◽  
Igor A Mikhailopulo
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Reaction Conditions ◽  
E Coli ◽  
No Formation ◽  
Structural Peculiarities ◽  
Substrate Properties ◽  
Nucleoside Phosphorylases ◽  
Derivatives Of

The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.

Myriad post-Myriad

2020 ◽  
Author(s):  
Shubha Ghosh
Keyword(s):  
Dna Sequences ◽  
Negative Impact ◽  
Genetic Data ◽  
Brca1 And Brca2 ◽  
Minimal Impact ◽  
Synthetic Dna ◽  
The Us ◽  
Myriad Genetics ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Primers

Abstract The US Supreme Court’s 2013 decision, holding patent claims to isolated, endogenous deoxyribonucleic acid (DNA) sequences to be invalid, seemed to have limited negative impact on Myriad Genetics whose patent on the isolated BRCA1 and BRCA2 genes were at the heart of the case. This article explains this minimal impact in two ways. First, the Court’s decision still left synthetic DNA patentable, leaving that as a fruitful source for commercialization by companies like Myriad. The Federal Circuit’s subsequent decision, however, invalidated Myriad’s product claims over the synthetic polymerase chain reaction primers based on the isolated DNA sequences. Second, the Court’s decision did not address the patentability of mined genetic data for diagnostic and therapeutic purposes. This field of genetic data mining is precisely where Myriad has moved in its patenting activity.

scholarly journals MESA: automated assessment of synthetic DNA fragments and simulation of DNA synthesis, storage, sequencing and PCR errors

2020 ◽  
Vol 36 (11) ◽  
pp. 3322-3326
Author(s):  
Michael Schwarz ◽  
Marius Welzel ◽  
Tolganay Kabdullayeva ◽  
Anke Becker ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Web Application ◽  
De Novo ◽  
Supplementary Information ◽  
Dna Fragments ◽  
Synthetic Dna ◽  
Custom Made ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Error Probabilities

Abstract Summary The development of de novo DNA synthesis, polymerase chain reaction (PCR), DNA sequencing and molecular cloning gave researchers unprecedented control over DNA and DNA-mediated processes. To reduce the error probabilities of these techniques, DNA composition has to adhere to method-dependent restrictions. To comply with such restrictions, a synthetic DNA fragment is often adjusted manually or by using custom-made scripts. In this article, we present MESA (Mosla Error Simulator), a web application for the assessment of DNA fragments based on limitations of DNA synthesis, amplification, cloning, sequencing methods and biological restrictions of host organisms. Furthermore, MESA can be used to simulate errors during synthesis, PCR, storage and sequencing processes. Availability and implementation MESA is available at mesa.mosla.de, with the source code available at github.com/umr-ds/mesa_dna_sim. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Tracing Septic Pollution Sources Using Synthetic DNA Tracers: Proof of Concept

2019 ◽  
Vol 12 ◽  
pp. 117862211986379 ◽  
Author(s):  
Christine B Georgakakos ◽  
Paul L Richards ◽  
M Todd Walter
Keyword(s):  
Preferential Flow ◽  
Quantitative Polymerase Chain Reaction ◽  
Breakthrough Curves ◽  
Septic Systems ◽  
Proof Of Concept ◽  
Septic System ◽  
Flow Paths ◽  
Synthetic Dna ◽  
Preferential Flow Paths ◽  
Polymerase Chain

Contamination from septic systems is one of the most difficult sources of nonpoint source (NPS) pollution to quantify. Quantification is difficult in part because locating malfunctioning septic systems within a watershed is challenging. This study used synthetic-DNA-based tracers to track flows from 2 septic systems. Sample DNA was quantified using quantitative polymerase chain reaction (qPCR). This technology could be especially useful for simultaneously assessing multiple septic systems because there are essentially infinite unique combinations of DNA bases such that unique tracers could be engineered for each septic system. Two studies were conducted: the first, to determine whether the tracers move through septic systems (experiment 1), and the second, to determine whether the tracers were detectable at watershed scales (experiment 2). In both cases, clear, although complex, breakthrough curves were detected. Experiment 1 revealed possible preferential flow paths that might not have been otherwise obvious, indicative of short circuiting systems. This proof of concept suggests that these tracers could be applied to watersheds suspected of experiencing NPS septic system pollution.

Pyrophosphoryl Derivatives of 1-(2-Deoxy-3-O-Phosphonomethyl-β-and-α-D-Erythro-Pentofuranosyl)Thymine: Synthesis and Substrate Properties Towards Some DNA Polymerases

2000 ◽  
Vol 19 (10-12) ◽  
pp. 1885-1909 ◽  
Author(s):  
Igor A. Mikhailopulo ◽  
Tamara I. Kulak ◽  
Olga V. Tkachenko ◽  
Svetlana L. Sentyureva ◽  
Lyubov S. Victorova ◽  
...  
Keyword(s):  
Dna Polymerases ◽  
Substrate Properties ◽  
Derivatives Of

scholarly journals Template-Directed Nonenzymatic Primer Extension Using 2-Methylimidazole-Activated Morpholino Derivatives of Guanosine and Cytidine

2019 ◽  
Vol 141 (30) ◽  
pp. 12159-12166 ◽  
Author(s):  
Weicheng Zhang ◽  
Ayan Pal ◽  
Alonso Ricardo ◽  
Jack W. Szostak
Keyword(s):  
Primer Extension ◽  
Derivatives Of
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