Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor–donor–acceptor and acceptor–acceptor–donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells

Research on the Intelligent Safety Monitoring System of Pipeline Corrosion in Acidic Oil and Gas Fields—II

AbstractAll kinds of corrosion monitoring techniques have their own advantages and obvious disadvantages when applied in acidic oil and gas fields. According to the characteristics of various technologies, an intelligent safety monitoring system was established based on electrochemical noise probe with galvanic corrosion probe, electrochemical hydrogen permeation probe and electric resistance probe. This paper presents the development of monitoring unit, system integration, and field test and data analysis. The results demonstrate that electrochemical noise not only determines the occurrence of corrosion, but also shows the characteristic of localized corrosion clearly; electrochemical hydrogen permeation technique reveals several advantages in the monitoring progress including simplicity, high sensitivity and high reliability, while the improved electric resistance probe shows a better environmental suitability. The accuracy and reliability of corrosion monitoring has been greatly increased by this integrated technique which can achieve the consistency and complementation of much information

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern

To support efforts to develop a ‘synthetic biology’ based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson–Crick complement, the purine analog 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where ‘py’ indicates a pyrimidine analog and ‘pu’ indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA

One example of an ‘artifically expanded genetic information system’ (AEGIS)

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> Nucleobase pairing in this system conforms to the Watson–Crick geometry, with large purines (or purine analogs, both indicated by ‘pu’) pairing with small pyrimidines (or pyrimidine analogs, both indicated by ‘py’). The hydrogen-bonding acceptor (A) and donor (D) groups are listed from the major to the minor groove as indicated. The heterocycles shown are current implementations of the indicated hydrogen-bonding patterns; others are conceivable. Unshared pairs of electrons (or ‘electron density’) presented to the minor groove are shown by the shaded lobes. The nucleotides implementing the pyDDA:puAAD hydrogen-bonding pattern, the subject of this article, is at the bottom right

() Extension of primers using the indicated diastereoisomers of dPTPαS with and 9°N (modified) DNA polymerases for the times indicated

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> The positions of migration of the unextended primer, primer extended by 1 nt and the primer extended by 2 nt, are indicated by P(N), N + 1, N + 2, respectively. The amount of oligonucleotide loaded to mark the position where the primer runs (lane 1) was less than for other lanes in the gel; () Exo III digestion of the products of 2-min primer extension reactions using the indicated diastereomers (S or R) of alpha-thio-dPTP (from a). Each primer extension product was purified with a QIAquick column and digested with 20 Units of Exo III for the indicated times. The control shows the degradation of 5′-P-labeled primer Z-SS-S19 (25mer) in a duplex with unlabeled Z-51-Temp, establishing that these are degraded by Exo III

Exo III digestion of single- and double-stranded DNA containing phosphorothioate linkages

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> Denaturing (7 M urea) PAGE showing digestion of double-stranded substrate (the duplex between 5′-P-labeled G*-2S-51 or C*-2S-51 and the complementary C-51-Temp or G-51-Temp), and single-stranded substrate (5′-P-labeled G*-2S-51 or C*-2S-51). () Digestion with high concentrations of Exo III (0.5 or 2.5 U/µl, as indicated) for the indicated times. For dsDNA substrates, the ratio of G*-2S-51/C-51-Temp or C*-2S-51/G-51-Temp was 1/1 or 1/1.5 as indicated. () Digestion with low concentrations of Exo III (0.025 and 0.125 U/µl, as indicated) for the indicated times. The loading of the substrate 51mer was reduced to prevent overloading of the gel; thus, the absolute intensities of these bands cannot be compared with the intensities in other lanes in the gel