Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences Single Molecule Real-Time sequencing. By employing reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second strand synthesis. Deletion and insertion rates increase for all RTs during second strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Programmable cleavage of linear double-stranded DNA by combined action of Argonaute CbAgo from Clostridium butyricum and nuclease deficient RecBC helicase from E. coli.

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Currently, the use of mesophilic pAgos as programmable endonucleases is hampered by their limited action on double-stranded DNA (dsDNA). We demonstrate here that efficient cleavage of linear dsDNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated in vitro via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.03-25 kb dsDNAs. When combined with RecBexo-C, CbAgo could cleave targets located 11-12.5 kb from the ends of linear dsDNA at 37°C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate DNA fragments with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. The combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for in vitro use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of linear dsDNA at any desired location.

Mismatch discrimination and sequence bias during end-joining by DNA ligases.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3'-OH and 5'-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

Brillouin-Scattering Induced Noise in DAS: A Case Study.

In the paper, the effect of spontaneous Brillouin scattering (SpBS) is analyzed as a noise source in distributed acoustic sensors (DAS). The intensity of the SpBS wave fluctuates over time, and these fluctuations increase the noise power in DAS. Based on experimental data, the probability density function (PDF) of the spectrally selected SpBS Stokes wave intensity is negative exponential, which corresponds to the known theoretical conception. Based on this statement, an estimation of the average noise power induced by the SpBS wave is given. This noise power equals the square of the average power of the SpBS Stokes wave, which in turn is approximately 18 dB lower than the Rayleigh backscattering power. The noise composition in DAS is determined for two configurations, the first for the initial backscattering spectrum and the second for the spectrum in which the SpBS Stokes and anti-Stokes waves are rejected. It is established that in the analyzed particular case, the SpBS noise power is dominant and exceeds the powers of the thermal, shot, and phase noises in DAS. Accordingly, by rejecting the SpBS waves at the photodetector input, it is possible to reduce the noise power in DAS. In our case, this rejection is carried out by an asymmetric Mach-Zehnder interferometer (MZI). The rejection of the SpBS wave is most relevant for broadband photodetectors, which are associated with the use of short probing pulses to achieve short gauge lengths in DAS.

Selenium Dibromide Click Chemistry: The Efficient Synthesis of Novel Selenabicyclo[3.3.1]nonene-2 and -nonane Derivatives.

Highly efficient and convenient methods for the preparation of 35 novel derivatives of 9-selenabicyclo[3.3.1]nonane and 9-selenabicyclo[3.3.1]nonene-2 in high yields based on the adduct of the transannular addition of SeBr2 to 1,5-cyclooctadiene were developed. The methods for the amination of the adduct made it possible to obtain both diamino selenabicyclo[3.3.1]nonane derivatives and their dihydrobromide salts in one step in 88-98% yields. The methods meet the criteria of click chemistry. Compounds with high glutathione peroxidase mimetic activity were found among water-soluble dihydrobromide salts. The selective reaction of 2,6-dibromo-9-selenabicyclo[3.3.1]nonane with acetonitrile to form 6-bromo-9-selenabicyclo[3.3.1]nonene-2 was discovered. The latter compound served as a promising starting material to give rise to the new class of selenabicyclo[3.3.1]nonene-2 derivatives, e.g., 6-alkoxy-9-selenabicyclo[3.3.1]nonenes were obtained in 94-99% yields.

A Cost-Effective Distributed Acoustic Sensor for Engineering Geology.

A simple and cost-effective architecture of a distributed acoustic sensor (DAS) or a phase-OTDR for engineering geology is proposed. The architecture is based on the dual-pulse acquisition principle, where the dual probing pulse is formed via an unbalanced Michelson interferometer (MI). The necessary phase shifts between the sub-pulses of the dual-pulse are introduced using a 3 × 3 coupler built into the MI. Laser pulses are generated by direct modulation of the injection current, which obtains optical pulses with a duration of 7 ns. The use of an unbalanced MI for the formation of a dual-pulse reduces the requirements for the coherence of the laser source, as the introduced delay between sub-pulses is compensated in the fiber under test (FUT). Therefore, a laser with a relatively broad spectral linewidth of about 1 GHz can be used. To overcome the fading problem, as well as to ensure the linearity of the DAS response, the averaging of over 16 optical frequencies is used. The performance of the DAS was tested by recording a strong vibration impact on a horizontally buried cable and by the recording of seismic waves in a borehole in the seabed.

Click Chemistry of Selenium Dihalides: Novel Bicyclic Organoselenium Compounds Based on Selenenylation/Bis-Functionalization Reactions and Evaluation of Glutathione Peroxidase-like Activity.

A number of highly efficient methods for the preparation of novel derivatives of 9-selenabicyclo[3.3.1]nonane in high yields based on selenium dibromide and cis,cis-1,5-cyclooctadiene are reported. The one-pot syntheses of 2,6-diorganyloxy-9-selenabicyclo[3.3.1]nonanes using various O-nucleophiles including alkanols, phenols, benzyl, allyl, and propargyl alcohols were developed. New 2,6-bis(1,2,3-triazol-1-yl)-9-selenabicyclo[3.3.1]nonanes were obtained by the copper-catalyzed 1,3-dipolar cycloaddition of 2,6-diazido-9-selenabicyclo[3.3.1]nonane with unsubstituted gaseous acetylene and propargyl alcohol. The synthesis of 2,6-bis(vinylsulfanyl)-9-selenabicyclo[3.3.1]nonane, based on the generation of corresponding dithiolate anion from bis[amino(iminio)methylsulfanyl]-9-selenabicyclo[3.3.1]nonane dibromide, followed by the nucleophilic addition of the dithiolate anion to unsubstituted acetylene, was developed. The glutathione peroxidase-like activity of the obtained water-soluble products was estimated and compounds with high activity were found. Overall, 2,6-Diazido-9-selenabicyclo[3.3.1]nonane exhibits the highest activity among the obtained compounds.

Recent Advances in Design and Synthesis of Diselenafulvenes, Tetraselenafulvalenes, and Their Tellurium Analogs and Application for Materials Sciences.

The first organic metals were obtained based on tetrathiafulvalene. The most significant advance in the field of organic metals was the discovery of superconductivity. The first organic superconductors were obtained based on tetramethyltetraselenafulvalene. These facts demonstrate great importance of tetraselenafulvalenes and their precursors, diselenafulvenes, for materials sciences. Derivatives of 1,4-diselenafulvene and 1,4,5,8-tetraselenafulvalene are useful building blocks for organic synthesis and donor units for the preparation of charge-transfer complexes and radical ion salts, the construction of organic metals, superconductors, organic Dirac materials, semiconductors, ferromagnets, and other conductive materials. This review covers the literature on the design, synthesis, and application of 1,4,5,8-tetraselenafulvalenes and 1,4-diselenafulvenes and their tellurium analogs over the past 15-20 years. These two classes of compounds are interconnected, since the main part of methods for the synthesis of tetraselenafulvalenes is based on the diselenafulvene derivatives as starting compounds. Special attention is paid to the development of novel efficient synthetic approaches to these classes of compounds. Conducting properties and distinguishing features of materials based on tetraselenafulvalenes and their tellurium analogs as well as examples of materials with high conductivity are discussed.

Selenium Dihalides Click Chemistry: Highly Efficient Stereoselective Addition to Alkynes and Evaluation of Glutathione Peroxidase-Like Activity of Bis(E-2-halovinyl) Selenides.

Highly efficient stereoselective syntheses of novel bis(E-2-chlorovinyl) selenides and bis(E-2-bromovinyl) selenides in quantitative yields by reactions of selenium dichloride and dibromide with alkynes were developed. The reactions proceeded at room temperature as anti-addition giving products exclusively with (E)-stereochemistry. The glutathione peroxidase-like activity of the obtained products was estimated and compounds with high activity were found. The influence of substituents in the products on their glutathione peroxidase-like activity was discussed.

Efficient Synthesis of a New Family of 2,6-Disulfanyl-9-selenabicyclo[3.3.1]nonanes.

The efficient synthesis of a new family of 2,6-disulfanyl-9-selenabicyclo[3.3.1]nonanes in high yields has been developed based on 9-selenabicyclo[3.3.1]nonane-2,6-dithiolate anion generated from bis-isothiouronium salt of 2,6-dibromo-9-selenabicyclo[3.3.1]nonane. The derivatives of 2,6-disulfanyl-9-selenabicyclo[3.3.1]nonane containing alkyl, allyl and benzyl moieties have been prepared in 90-99% yields by nucleophilic substitution of 9-selenabicyclo[3.3.1]nonane-2,6-dithiolate anion with alkyl, allyl and benzyl halides. The reaction of nucleophilic addition of 9-selenabicyclo[3.3.1]nonane-2,6-dithiolate anion to alkyl propiolates afforded 2,6-di(vinylsulfanyl)-9-selenabicyclo[3.3.1]nonanes. The conditions for regio- and stereoselective addition of 9-selenabicyclo[3.3.1]nonane-2,6-dithiolate anion to a triple bond of alkyl propiolates have been found. To date, not a single representative of 2,6-disulfanyl-9-selenabicyclo[3.3.1]nonanes has been described in the literature.

A Novel Family of [1,4]Thiazino[2,3,4-ij]quinolin-4-ium Derivatives: Regioselective Synthesis Based on Unsaturated Heteroatom and Heterocyclic Compounds and Antibacterial Activity.

A novel family of [1,4]thiazino[2,3,4-ij]quinolin-4-ium derivatives was synthesized by annulation reactions of 8-quinolinesulfenyl chloride with unsaturated heteroatom and heterocyclic compounds. It was found that the reactions with 4-pentenoic and 5-hexenoic acids, allyl chloride and bromide, allyl cyanate and vinyl heterocyclic compounds (N-vinyl pyrrolidin-2-one and 1-vinylimidazole) proceeded in a regioselective mode but with the opposite regiochemistry. The reactions with vinyl heterocyclic compounds included electrophilic addition of the sulfur atom of 8-quinolinesulfenyl chloride to the ß-carbon atom of the vinyl group. In the case of other substrates, the annulation proceeded with the attachment of the sulfur atom to the α-carbon atom of the vinyl group. The antibacterial activity of novel water-soluble compounds against Enterococcus durans, Bacillus subtilis and Escherichia coli was evaluated. Compounds with high antibacterial activity were found.

Efficient Regioselective Synthesis of Novel Condensed Sulfur-Nitrogen Heterocyclic Compounds Based on Annulation Reactions of 2-Quinolinesulfenyl Halides with Alkenes and Cycloalkenes.

The preparation of novel reagents 2-quinolinesulfenyl chloride and bromide based on available 2-mercaptoquinoline has been described. This approach opens up opportunities for the introduction of 2-quinolinesulfenyl chloride and bromide into organic synthesis. Regioselective synthesis of novel 1,2-dihydro[1,3]thiazolo[3,2-a]quinolin-10-ium derivatives in high yields has been developed by annulation reactions of 2-quinolinesulfenyl chloride and bromide with alkenes. Condensed tetracyclic products have been obtained by the reactions of 2-quinolinesulfenyl chloride and bromide with cycloalkenes. The opposite regiochemistry in the reactions with styrene, isoeugenol and 1-alkenes was discussed.

Efficient Regioselective Synthesis of Novel Water-Soluble 2H,3H-[1,4]thiazino[2,3,4-ij]quinolin-4-ium Derivatives by Annulation Reactions of 8-quinolinesulfenyl Halides.

Regioselective synthesis of novel 2H,3H-[1,4]thiazino[2,3,4-ij]quinolin-4-ium derivatives has been developed by annulation reactions of 8-quinolinesulfenyl halides with vinyl chalcogenides (vinyl ethers, divinyl sulfide, divinyl selenide and phenyl vinyl sulfide) and tetravinyl silane. The novel reagent 8-quinolinesulfenyl bromide was used in the annulation reactions. The influence of the substrate structure and the nature of heteroatoms on the direction of the reactions and on product yields has been studied. The opposite regiochemistry was observed in the reactions with vinyl chalcogenides and tetravinyl silane. The obtained condensed heterocycles are novel water-soluble functionalized compounds with promising biological activity.

Quantum Chemical and Experimental Studies of an Unprecedented Reaction Pathway of Nucleophilic Substitution of 2-Bromomethyl-1,3-thiaselenole with 1,3-Benzothiazole-2-thiol Proceeding Stepwise at Three Different Centers of Seleniranium Intermediates.

The results of quantum chemical and experimental studies of the reaction of 2-bromomethyl-1,3-thiaselenole with 1,3-benzothiazole-2-thiol made it possible to discover the unprecedented pathway of this reaction, which proceeds stepwise at three different centers of seleniranium intermediates. The first stage includes an attack of thiolate anion at the selenium atom of the seleniranium cation accompanied by ring opening with the formation of (Z)-2-[(1,3-benzothiazol-2-ylsulfanyl)selanyl]ethenyl vinyl sulfide, which is converted to six-membered heterocycle, 2-(2,3-dihydro-1,4-thiaselenin-2-ylsulfanyl)-1,3-benzothiazole, in a 99% yield. The latter compound undergoes rearrangement with ring contraction producing five-membered heterocycle, 2-[(1,3-thiaselenol-2-ylmethyl)sulfanyl]-1,3-benzothiazole, in a 99% yield (the thermodynamic product). The formation of 1,2-bis[(Z)-2-(vinylsulfanyl)ethenyl] diselenide is the result of the disproportionation of (Z)-2-[(1,3-benzothiazol-2-ylsulfanyl)selanyl]ethenyl vinyl sulfide. Thus, based on the quantum chemical and experimental studies, a regioselective synthesis of the reaction products in high yields was developed.

A Regioselective Synthesis of Novel Functionalized Organochalcogen Compounds by Chalcogenocyclofunctionalization Reactions Based on Chalcogen Halides and Natural Products.

The regioselective synthesis of novel functionalized condensed organochalcogen compounds by chalcogenocyclofunctionalization reactions based on chalcogen halides and the natural products thymol and carvacrol has been developed. The reactions of selenium dibromide with allyl thymol and allyl carvacrol proceeded in methylene chloride at room temperature in the presence of NaHCO3 affording bis[(7-isopropyl-4-methyl-2,3-dihydro-1-benzofuran-2-yl)methyl] and bis[(4-isopropyl-7-methyl-2,3-dihydro-1-benzofuran-2-yl)methyl] selenides in 90-92% yield. Similar sulfides were obtained in 70-72% yields by the reaction of sulfur dichloride in chloroform under reflux. Trihalotellanes containing the same organic moieties were synthesized from allyl thymol, allyl carvacrol and tellurium tetrachloride or tetrabromide in quantitative yields. Corresponding functionalized ditellurides were prepared in 91-92% yields by the reduction of the trichlorotellanes with sodium metabisulfite in two-phase solvent system. The comparison of reactivity of sulfur, selenium and tellurium halides in chalcogenocyclofunctionalization and distinguishing features of each reaction were discussed.

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

(Z,Z)-Selanediylbis(2-propenamides): Novel Class of Organoselenium Compounds with High Glutathione Peroxidase-Like Activity. Regio- and Stereoselective Reaction of Sodium Selenide with 3-Trimethylsilyl-2-propynamides.

The efficient regio- and stereoselective synthesis of (Z,Z)-3,3'-selanediylbis(2-propenamides) in 76-93% yields was developed based on the reaction of sodium selenide with 3-trimethylsilyl-2-propynamides. (Z,Z)-3,3'-Selanediylbis(2-propenamides) are a novel class of organoselenium compounds. To date, not a single representative of 3,3'-selanediylbis(2-propenamides) has been described in the literature. Studying glutathione peroxidase-like properties by a model reaction showed that the activity of the obtained products significantly varies depending on the organic moieties in the amide group. Divinyl selenide, which contains two lipophilic cyclohexyl substituents in the amide group, exhibits very high glutathione peroxidase-like activity and this compound is considerably superior to other products in this respect.

Natural Compounds and Their Structural Analogs in Regio- and Stereoselective Synthesis of New Families of Water-Soluble 2H,3H-[1,3]thia- and -Selenazolo[3,2-a]pyridin-4-ium Heterocycles by Annulation Reactions.

It has been found that both eugenol and isoeugenol derivatives reacted with 2-pyridinesulfenyl and 2-pyridineselenenyl halides in a regioselective mode affording products with opposite regiochemistry. Synthesis of new families of 2H,3H-[1,3]thia- and -selenazolo[3,2-a]pyridin-4-ium heterocycles has been developed by annulation reactions of 2-pyridinechalcogenyl halides with natural compounds (eugenol, isoeugenol, methyl eugenol, methyl isoeugenol, acetyl eugenol, trans-anethole) and their structural analogs. The influence of the substrate structure and the nature of halogen on the product yields are studied. The 2-pyridinesulfenyl and 2-pyridineselenenyl chlorides are more efficient reagents compared to corresponding bromides. The obtained condensed heterocycles are novel water-soluble functionalized compounds with promising biological activity.

Remarkable Alkene-to-Alkene and Alkene-to-Alkyne Transfer Reactions of Selenium Dibromide and PhSeBr. Stereoselective Addition of Selenium Dihalides to Cycloalkenes.

The original goal of this research was to study stereochemistry of selenium dihalides addition to cycloalkenes and properties of obtained products. Remarkable alkene-to-alkene and alkene-to-alkyne transfer reactions of selenium dibromide and PhSeBr were discovered during this research. The adducts of selenium dibromide with alkenes or cycloalkenes easily exchange SeBr2 with other unsaturated compounds, including acetylenes, at room temperature, in acetonitrile. Similar alkene-to-alkene and alkene-to-alkyne transfer reactions of the PhSeBr adducts with alkenes or cycloalkenes take place. The supposed reaction pathway includes the selenium group transfer from seleniranium species to alkenes or alkynes. It was found that the efficient SeBr2 and PhSeBr transfer reagents are Se(CH2CH2Br)2 and PhSeCH2CH2Br, which liberate ethylene, leading to a shift in equilibrium. The regioselective and stereoselective synthesis of bis(E-2-bromovinyl) selenides and unsymmetrical E-2-bromovinyl selenides was developed based on the SeBr2 and PhSeBr transfer reactions which proceeded with higher selectivity compared to analogous addition reactions of SeBr2 and PhSeBr to alkynes under the same conditions.