Optimization of interstrand hydrophobic packing interactions within unnatural DNA base pairs

As part of an effort to expand the genetic alphabet, we have evaluated a large number of predominantly hydrophobic unnatural base pairs. We now report the synthesis and stability of unnatural base pairs formed between simple phenyl rings modified at different positions with methyl groups. Surprisingly, several of the unnatural base pairs are virtually as stable as a natural base pair in the same sequence context. The results show that neither hydrogen-bonding nor large aromatic surface area are required for base pair stability within duplex DNA and that interstrand interactions between small aromatic rings may be optimized for both stability and selectivity. These smaller nucleobases are not expected to induce the distortions in duplex DNA or at the primer terminus that seem to limit replication of larger unnatural base pairs, and they therefore represent a promising approach to the expansion of the genetic alphabet.     

Optimization of unnatural base pair packing for polymerase recognition

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.     

Fast approximate methods for calculating nucleic acid base pair interaction energies

Interaction enthalpies for six base pairs have been computed at a variety of efficient levels of electronic structure theory and compared to experiment. In addition to previously defined levels of theory, modified Hamiltonians with adjusted parameters in hybrid Hartree-Fock/density functionals and semiempirical neglect-of-diatomic-differential-overlap models were examined. Of the pure and hybrid density functional levels, mPWPW91/MIDI! performed most satisfactorily, as judged by comparison not only to the available experimental data, but also to data from more robust electronic structure methods for 22 additional base pairs. The low computational cost of the mPWPW91/MIDI! model was further exploited in an investigation of various base trimers, tetramers, and one base pentamer. A carefully reparameterized semiempirical model, PM3(BP), was able to achieve similar levels of accuracy at a still greater savings in terms of computational effort.     

Hybrid one-electron/many-electron methods for ionized states of molecular clusters

To describe singly-ionized states of molecular clusters we devised an effective Hamiltonian approach that combines (1) accurate monomer ionization potentials from many-electron wave functions with (2) polarization shifts and (3) effective monomer couplings obtained from a simple one-electron approach (the superposition-of-fragment-states (SFS) method [Valeev et al., J. Am. Chem. Soc., 2006, 128, 9882]). The accuracy of the intermolecular coupling parameters evaluated with SFS Hartree-Fock (HF) and Density-Functional-Theory (DFT) variants was evaluated for several weakly-bound dimers and compared against the state-of-the-art equation-of-motion ionization-potential coupled-cluster singles and doubles (EOM-IP-CCSD) data of Krylov and co-workers. The SFS-HF method produces coupling integrals accurate to a few percent, whereas SFS-DFT predictions are substantially worse. A hybrid approach combining SFS-HF couplings and shifts with EOM-IP-CCSD ionization potentials of monomers (denoted as SFS-EOM-IP-CCSD) was applied to ionized states of two conformers of a benzene dimer and ten representative DNA base pairs. The 16 considered SFS-EOM-IP-CCSD ionization potentials of the benzene dimer differed from the reference EOM-IP-CCSD IPs of Krylov and co-workers [Pieniazek et al., J. Chem. Phys. 2007, 127, 044317; Bravaya et al., Phys. Chem. Chem. Phys. 2010, 12, 2261] by less than 0.1 eV on average, and at most by 0.2 eV. For the DNA base pairs the mean absolute (median) deviation of the SFS-EOM-IP-CCSD IPs was 0.27 (0.23) eV; several deviations for non-Koopmans states were as large as 0.9 eV. The SFS-EOM-IP-CCSD method can be readily applied to large molecular clusters with computational effort scaling cubically with the size of the cluster.     

Multiple automatic base selection: Protein–ligand docking based on incremental construction without manual intervention

A possible way of tackling the molecular docking problem arising in computer- aided drug design is the use of the incremental construction method. This method consists of three steps: the selection of a part of a molecule, a so- called base fragment, the placement of the base fragment into the active site of a protein, and the subsequent reconstruction of the complete drug molecule. Assuming that a part of a drug molecule is known, which is specific enough to be a good base fragment, the method is proven to be successful for a large set of docking examples. In addition, it leads to the fastest algorithms for flexible docking published so far. In most real-world applications of docking, large sets of ligands have to be tested for affinity to a given protein. Thus, manual selection of a base fragment is not practical. On the other hand, the selection of a base fragment is critical in that only few selections lead to a low-energy structure. We overcome this limitation by selecting a representative set of base fragments instead of a single one. In this paper, we present a set of rules and algorithms to automate this selection. In addition, we extend the incremental construction method to deal with multiple fragmentations of the drug molecule. Our results show that with multiple automated base selection, the quality of the docking predictions is almost as good as with one manually preselected base fragment. In addition, the set of solutions is more diverse and alternative binding modes with low scores are found. Although the run time of the overall algorithm increases, the method remains fast enough to search through large ligand data sets.     

Boosting the advantages of biosensors: Niche applicability and fitness for environmental purpose

Environmental assessments rely on data derived from metrological schemes in order to draw in due detail the state of the ecosystems. The reliability of the data and the cost-effectiveness of the scheme become critical, especially if the ultimate goal involves decision making and policy development. Biosensor platforms provide the versatility required for integrating environmental, metrological and operational parameters at the design phase. The pertinency and the opportunity for environmental biosensing is apparent, although not seized appropriately with only a few environmental biosensors in the market. This study is based on a technology forecasting approach using bibliometric data to trace the dynamics of the science base and content analysis to identify the drawbacks of the technology base. The results are compared with market forecasts in an effort to align research dynamics with market opportunities. A number of key findings emerged, such as the increase in networking and the establishment of a multi-disciplinarity trend, as well as a shifting of research scope towards ready-to-market designing. Notwithstanding, a renewed interest of the physical chemistry sector has been identified herein, indicating necessary revisions of the science-base, adding a critical parameter in the uncertainty of trajectories.     

Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues

In an effort to develop an orthogonal third base pair for the storage of genetic information, thiophene and furan heterocycles have been examined as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases. These results expand the potential nucleobase analogues with which the genetic alphabet may be expanded to include five-membered-ring heterocycles.     

Chemical modelling of multicomponent mixtures: quality assurance is more than just equilibrium data quality assessment

Despite the large amount of data available, the great effort put into searches for the ??best?? parameters and many comparative modelling studies, considerable uncertainties continue to plague chemical thermodynamics. An important factor in this ongoing failure has been the notion that the problem can be solved by better assessment of data quality on a case-by-case basis. This approach has proved strikingly unsuccessful. A different methodology must therefore be found to meet the general requirements of thermodynamic modelling in aquatic chemistry. This paper discusses current practices in quality assessment of thermodynamic data and the problems associated with them. It outlines a general approach which might address the above problem based on two concepts: (i) using large databases to store as much of the available data as possible in the form that it appears in the literature along with an assessed ??score?? or ??measure of information content?? and (ii) then using automatic mechanisms informed by this score to produce the thermodynamically consistent datasets needed for modelling calculations.     

Separation performance of single-stranded DNA electrophoresis in photopolymerized cross-linked polyacrylamide gels

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models.     

A scalable approach to combinatorial library design for drug discovery

In this paper, we propose an algorithm for the design of lead generation libraries required in combinatorial drug discovery. This algorithm addresses simultaneously the two key criteria of diversity and representativeness of compounds in the resulting library and is computationally efficient when applied to a large class of lead generation design problems. At the same time, additional constraints on experimental resources are also incorporated in the framework presented in this paper. A computationally efficient scalable algorithm is developed, where the ability of the deterministic annealing algorithm to identify clusters is exploited to truncate computations over the entire data set to computations over individual clusters. An analysis of this algorithm quantifies the tradeoff between the error due to truncation and computational effort. Results applied on test data sets corroborate the analysis and show improvement by factors as large as 10 or more, depending on the data sets.     

Evaluation of energy-dispersive x-ray spectra with the aid of expert systems

Two recently developed Expert systems in the field of x-ray fluorescence are discussed. The first deals with the qualitative interpretation of x-ray spectra; it is characterized by a large knowledge base and a relatively small data base, both of which are of uniform structure. The second contains knowledge on quantitative spectrum evaluation and so has a much larger data base; its knowledge base is much smaller (at present), but contains several different types of rules.     

A functionally flexible interatomic energy function based on classical potentials

A flexible potential energy function is proposed herein on the basis of classical potential functions of Lennard-Jones, Morse, Buckingham and Linnett. By introducing two indices, which range from 0 to 1, the proposed function can either be reduced to the classical potentials by fixing the indices at their extremes or be used as a flexible function for curve-fitting interatomic energy data over a broad range of interatomic distance. In a departure from previous effort that give exact parametric relationships only at the minimum well-depth, the present paper quantitatively accounts for the discrepancies at very large interatomic compression and stretching.     

All-trans-N-retinylidenetryptamine Schiff base in surfactant solubilized water pools in heptane—a fluorescence study

All-trns-N-retinylidenetryptamine Schiff base was incorporated into aerosol-OT (AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/heptane reverse micelles. This micellar system was used as a model to study the retinal-tryptophan interactions in retinal proteins. The retinylidene Schiff base remains stable in the presence of reverse micelle-solubilized water pools. Partition coefficient and microviscosity measurements show that the Schiff base is located in the micellar interphase. The results are discussed in terms of the interaction between the retinylidene chromophore and the active site environment of rhodopsin and bacteriorhodopsin. In the present model, the quencher and emitting units are covalently attached, and are separated by two carbon spacer units. The fluorescence emission data obtained for the micelle-intercalated Schiff base chromophore are compared with the fluorescence of the native protein and intermediates in the photochemical cycle of bacteriofhodopsin. A comparison of the data obtained for tryptamine and the Schiff base with the results available for bacteriorhodopsin and bacterioopsin reveals that there is a large degree of quenching on intercalation of the retinylidene chromophore in the vicinity of the fluorophore. Evidence provided by this model suggests that energy transfer to retinal can occur from tryptophan residues located in the retinal pocket in the native protein. Thus the retinylidene unit can act as a quencher of the energy of tryptophan, the nature and extent of which may depend on the conformation and relative orientation of the protein-bound fluorophore.     

Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs

Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.     

Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs

Expansion of the genetic alphabet with an unnatural base pair is a long‐standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d 5SICS and analogues of d MMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new d MMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d 5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, d NaM –d 5SICS , to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure–activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (d NMO1 ) that is a better partner for d 5SICS than any of the previously identified d MMO2 analogues with the exception of d NaM . We also found that d NaM –d 5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.     

Array files for computational chemistry: MP2 energies

A simple message‐passing implementation for distributed disk storage, called array files (AF), is described. It is designed primarily for parallelizing computational chemistry applications but it should be useful for any application that handles large amounts of data stored on disk. AF allows transparent distributed storage and access of large data files. An AF consists of a set of logically related records, i.e., blocks of data. It is assumed that the records have the typical dimension of matrices in quantum chemical calculations, i.e., they range from 0.1 to ～32 MB in size. The individual records are not striped over nodes; each record is stored on a single node. As a simple application, second‐order Møller‐Plesset (MP2) energies have been implemented using AF. The AF implementation approaches the efficiency of the hand‐coded program. MP2 is relatively simple to parallelize but for more complex applications, such as Coupled Cluster energies, the AF system greatly simplifies the programming effort. © 2007 Wiley Periodicals, Inc. J Comput Chem, 2007.     

The role of pattern recognition in the computer-aided classification of mass spectra

The requirements for the use of pattern recognition techniques as an aid in the identification of chemical substances from their mass spectra are reviewed. Decision-tree pattern recognition is recommended as potentially satisfying these requirements. Examples of this approach using a large data base of mass spectra are provided.     

Minor groove hydrogen bonds and the replication of unnatural base pairs

As part of an effort to expand the genetic alphabet, we examined the synthesis of DNA with six different unnatural nucleotides bearing methoxy-derivatized nucleobase analogues. Different nucleobase substitution patterns were used to systematically alter the nucleobase electronics, sterics, and hydrogen-bonding potential. We determined the ability of the Klenow fragment of E. coli DNA polymerase I to synthesize and extend the different unnatural base pairs and mispairs under steady-state conditions. Unlike other hydrogen-bond acceptors examined in the past, the methoxy groups do not facilitate mispairing, implying that they are not recognized by any of the hydrogen-bond donors of the natural nucleobases; however, they do facilitate replication. The more efficient replication results largely from an increase in the rate of extension of primers terminating at the unnatural base pair and, interestingly, requires that the methoxy group be at the ortho position where it is positioned in the developing minor groove and can form a functionally important hydrogen bond with the polymerase. Thus, ortho methoxy groups should be generally useful for the effort to expand the genetic alphabet.     

Synthesis,physicochemical elucidation,biological screening and molecular docking studies of a Schiff base and its metal(II) complexes

A Schiff base 1-((3-nitrophenylimino)methyl)naphthalen-2-olate (HL) and its two novel complexes with Zn(II) and Co(II) metals were successfully synthesized and characterized by FTIR, ¹H NMR, ¹³C NMR, elemental analysis, magnetic susceptibility, TGA and EIS-MS. Crystal of Schiff base was also characterized by X-ray analysis and experimental parameters were found in line with the theoretical parameters. Quantum mechanical approach was also used to compare structural and calculated parameters and to ensure the geometry of metal complexes. The photometric behaviors of all the synthesized compounds were investigated in a wide pH range using BR buffers. Appearance of isosbestic point suggested the existence of Schiff base molecules in different tautomeric forms. Binding of synthesized complexes with calf thymus DNA was explored by photometric and voltammetric titrations and binding constants were calculated. The results indicated that ligand and its metal complexes bind to DNA by intercalation mode. Docking studies indicate their binding possibilities with topoisomerase II. Moreover, all these prepared compounds were screened for enzyme inhibition, antibacterial, cytotoxic and in vivo antidiabetic activities and found active against one or other activity. This effort just provides preliminary data for some biological properties and which can act as foundation stone for their application in drug development.     

Characterization of Major and Minor Organic Pollutants in Wastewaters from Coal Gasification Processes

Abstract

In order to investigate the feasibility of anaerobic biological treatment for wastewaters generated from thermal gasification processes of coal, a characterization program was implemented whose major effort consisted in the elucidation of specific organic constituents contained in the wastewater. Solvent extraction in acid and base conditions followed by glass capillary gas chromatography in combination with several detectors (i.e., FID, NPD, and MS-DS) were employed for the investigation of major and minor “extractable” organic constituents. Direct aqueous injection on a polar glass capillary column (i.e., OV-351) was used for the major “nonsolvent extractable” organic constituents amenable to GC. The identity of 28 organic compounds was confirmed by comparison with pure standards. Phenol, the three cresol isomers, 5,5-dimethyl-hydantoin and 5-methyl,5-ethylhydantoin were identified as major wastewater constituents. Several substituted phenols (e.g., methyl, dimethyl, trimethyl, methylethyl, hydroxy and methoxy), pyridines, anilines, quinolines, PAHs, dibenzofuran and aldehydes were either confirmed or tentatively identified as minor wastewater constituents. Although the organics identified did not account for the total organic content, which implies the presence of still unidentified highly polar compounds, the information was utilized to set a data base for monitoring the biological treatment operations. Process monitoring data indicated that several organics (i.e., 5,5-dimethyl-hydantoin, 5-methyl,5-ethylhydantoin, o-cresol, m-cresol and p-cresol) were only partially removed by the treatment process employed.