Lev N. Krasnoperov

Luminescent probes for ultrasensitive detection of nucleic acids.

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Thermodynamics of the multi-stage DNA lesion recognition and repair by formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence—stopped-flow pre-steady-state kinetics

Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10–30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van’t Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.

Reaction CH3 + CH3 → C2H6 Studied over the 292-714 K Temperature and 1-100 bar Pressure Ranges.

Reaction of recombination of methyl radicals, CH3 + CH3 → C2H6 (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 292-714 K temperature and 1-100 bar pressure ranges (bath gas He), very close to the high-pressure limit. Methyl radicals were produced by photolysis of acetone at 193.3 nm or in the reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N2O at 193.3 nm, with CH4, and subsequent reaction of OH with CH4. Temporal profiles of CH3 were recorded via absorption at 216.36 and 216.56 nm using a xenon arc lamp and a spectrograph. The absolute intensity of the photolysis light inside the reactor was determined by an accurate in situ actinometry based on the ozone formation in photolysis of N2O/O2/N2 mixtures. The rate constant of reaction 1 in the high-pressure limit has a negative temperature dependence: k1,inf = (5.66 ± 0.43) × 10(-11)(T/298 K)(-0.37) cm(3) molecule(-1) s(-1) (292-714 K).

Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

Pressure dependence of the reaction H+O2(+Ar)→HO2(+Ar) in the range 1–900 bar and 300–700 K

The reaction H+O2 (+Ar)→HO2 (+Ar) was studied in a high pressure flow cell in the bath gas argon at pressures between 1 and 900 bar and temperatures between 300 and 700 K. H atoms were generated by laser flash photolysis of NH3 at 193.3 nm, HO2 radicals were monitored by light absorption at 230 nm. The results are consistent with experimental low pressure rate constants k0=[Ar] 2.5×10−32(T/300 K)−1.3 cm6 molecule−2 s−1 and theoretical high pressure rate constants k∞=9.5×10−11(T/300 K)+0.44 cm3 molecule−1 s−1 from the literature. The intermediate falloff curve was found to be best represented by k/k∞=[x/(1+x)]Fcent1/(1+(a+logx)2/(N±ΔN)2) with x=k0/k∞, a≈0.3, N≈1.05, ΔN≈0.1 (+ΔN for (a+logx) 0), and Fcent(Ar)≈0.5 independent of the temperature. A comparison with literature data between 300 and 1200 K does not confirm major deviations from third order kinetics in earlier medium pressure experiments.

Study of Volatile Organic Compounds Destruction by Dielectric Barrier Corona Discharge

Abstract An experimental and mechanistic study on the destruction of Volatile Organic Compounds (VOCs: Methane, Methyl Chloride, Chlorobenzene, Toluene, Methyl Ethyl Ketone, 1-Pentene and Cyclohexene) in 0.21: 0.79 oxygen-nitrogen mixture “zero air” over the concentration range of 5-100000 ppm by dielectric barrier corona discharge plasma was performed. Tubular-flow, coaxial-wire, AC-powered dielectric barrier corona discharge reactors were used to determine the kinetics of destruction, reaction products and electric power requirements. High efficiency VOC destruction/removal was demonstrated. Efficiency of destruction (calculated per unit volume of air) increases with decreasing VOC concentration. At low VOC concentrations (5-100 ppm) the energy required for destruction is 0.001- 0.1 J cm-3. The kinetics of destruction was shown to follow (in a first approximation, for all VOCs studied but methane) the “pseudo-first order” law: Cout /Cin = exp (-Ev /E1v o xvoc), where Ev is the energy density, E1vo is energy per unit volume required to reduce the concentration of VOC to the e-1 level in a hypothetical mixture with the unit mole fraction of VOC, and Xvoc is the mole fraction of VOC. Plausible mechanisms of destruction are considered and compared with the experimental data. Purely “chemical” (free radical) mechanisms based on initial activation of VOC molecules in reactions with oxygen and nitrogen atoms, or hydroxyl radicals were rejected as they fail to explain the absolute efficiency of the observed destruction as well as the destruction in nitrogen and helium. Other possible mechanisms, including ones based on ion-molecular reactions, are discussed.

Microporous SiO2/Vycor membranes for gas separation

In this study, porous Vycor tubes with 40 A initial pore diameter were modified using low pressure chemical vapor deposition (LPCVD) of SiO 2 . Diethylsilane (DES) in conjunction with O 2 or N 2 O were used as precursors to synthesize the SiO 2 films. Both “single side” (reactants flowing on the same side of porous membrane) and “counterflow” (reactants flowing on both sides of porous membrane) reactant geometries have been investigated. The flow of H 2 , He, N 2 , Ar, and toluene (C 7 H 8 ) was monitored in situ after each deposition period. Membranes modified by the “single side” reactants geometry exhibited good selectivities between small and large molecules. However, cracking in these membranes after prolonged deposition limited the maximum achievable selectivity values. Higher selectivities and better mechanical stability were achieved with membranes produced using the “counterflow” reactants geometry. Pore narrowing rate was observed to increase with oxidant flow (O 2 or N 2 O). For membranes prepared using both oxidants, selectivities on the order of 1000: 1 were readily attained for H 2 and He over N 2 , Ar, and C 7 H 8 . As compared to O 2 , the use of N 2 O caused improvements in both the pore narrowing rate and N 2 : C 7 H 8 selectivity. Membranes prepared using the “counterflow” geometry showed no signs of degradation or cracking after thermal cycling.

Kinetics of the Reaction of CH3O2 Radicals with OH Studied over the 292-526 K Temperature Range.

Reaction of methyl peroxy radicals with hydroxyl radicals, CH3O2 + OH → CH3O + HO2 (1a) and CH3O2 + OH → CH2OO + H2O (1b) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 292-526 K temperature range and pressure 1 bar (bath gas He). Hydroxyl radicals were generated in the reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N2O at 193.3 nm, with H2O. Methyl peroxy radicals were generated in the reaction of methyl radicals, CH3, produced in the photolysis of acetone at 193.3 nm, and subsequent reaction of CH3 with O2. Temporal profiles of OH were monitored via transient absorption of light from a DC discharge H2O/Ar low-pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light was determined by accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The overall rate constant of the reaction is k1a+1b = (8.4 ± 1.7) × 10(-11)(T/298 K)(-0.81) cm(3) molecule(-1) s(-1) (292-526 K). The branching ratio of channel 1b at 298 K is less than 5%.

Disproportionation channel of self-reaction of hydroxyl radical, OH + OH → H2O + O, studied by time-resolved oxygen atom trapping.

The disproportionation channel of the self-reaction of hydroxyl radicals, OH + OH → H(2)O + O (1a) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 298-414 K temperature and 3-10 bar pressure ranges (bath gas He). To distinguish channel 1a from the recombination channel 1b, OH + OH → H(2)O(2) (1b), time-resolved trapping of oxygen atoms, produced in channel 1a, was used. The ozone produced in the reaction of oxygen atoms with molecular oxygen was measured using strong UV absorption at 253.7 nm. The results of this study (k(1a) = (1.38 ± 0.20) × 10(-12) (T/300)(-0.76) confirm the IUPAC recommended value of Bedjanian et al. (J. Phys. Chem. A1999, 103, 7017-7025), as well as the negative temperature dependence over the temperature range studied, and do not confirm the ca. 1.8 higher value obtained in the most recent study of Bahng et al. (J. Phys. Chem. A2007, 111, 3850-3861). The V-shaped temperature dependence of k(1a) based on combined current and previous studies in the temperature range of 233-2380 K is k(1a) = (5.1 exp(-T/190 K) + 0.30(T/300 K)(1.73)) × 10(-12) cm(3) molecule(-1) s(-1).

Rate Constants and Hydrogen Isotope Substitution Effects in the CH3 + HCl and CH3 + Cl2 Reactions

The kinetics of the CH3 + Cl2 (k2a) and CD3 + Cl2 (k2b) reactions were studied over the temperature range 188-500 K using laser photolysis-photoionization mass spectrometry. The rate constants of these reactions are independent of the bath gas pressure within the experimental range, 0.6-5.1 Torr (He). The rate constants were fitted by the modified Arrhenius expression, k2a = 1.7 x 10(-13)(T/300 K)(2.52)exp(5520 J mol(-1)/RT) and k2b = 2.9 x 10(-13)(T/300 K)(1.84)exp(4770 J mol(-1)/RT) cm(3) molecule(-1) s(-1). The results for reaction 2a are in good agreement with the previous determinations performed at and above ambient temperature. Rate constants of the CH3 + Cl2 and CD3 + Cl2 reactions obtained in this work exhibit minima at about 270-300 K. The rate constants have positive temperature dependences above the minima, and negative below. Deuterium substitution increases the rate constant, in particular at low temperatures, where the effect reaches ca. 45% at 188 K. These observations are quantitatively rationalized in terms of stationary points on a potential energy surface based on QCISD/6-311G(d,p) geometries and frequencies, combined with CCSD(T) energies extrapolated to the complete basis set limit. 1D tunneling as well as the possibility of the negative energies of the transition state are incorporated into a transition state theory analysis, an approach which also accounts for prior experiments on the CH3 + HCl system and its various deuterated isotopic substitutions [Eskola, A. J.; Seetula, J. A.; Timonen, R. S. Chem. Phys. 2006, 331, 26].