Binding of topotecan to a nicked DNA oligomer in solution

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.     

Restricted-access material HPLC method for simultaneous determination of carboxylate and lactone forms of topotecan in human serum

A simple restricted-access media (RAM) HPLC method for simultaneous determination of lactone and carboxylate forms of topotecan (TPT) in human serum was established. Using a RAM analytical column, serum samples were directly injected into the HPLC system. The eluted peaks of two forms of TPT were monitored with a fluorescence detector. The separation was completed in 18 min. The linear range was 15–1000 ng/mL, intra-day and inter-day variations being less than 7%. The kinetic equation was constructed according to the analytical results. The equation shows that the course of TPT lactone form converting to carboxylate form in human serum at 37°C follows a first-order kinetics. Concentration of each form at the moment of sampling was calculated by extrapolation. The article is published in the original.     

Conformational heterogeneity observed in simulations of a pyrene-substituted DNA

NMR studies previously carried out for a DNA system with a pyrene-substituted base did not observe NOEs involving the adenine located 5' to the pyrene, and thus the conformation of the adenine was poorly defined in the resulting family of refined structures. However, chemical shift data suggested that an AT base pair may be present. We have carried out fully unrestrained molecular dynamics simulations starting from several members of the family of structures, and these simulations support the existence of an AT base pair for this region. Simulations in both explicit and implicit solvent were carried out, with each converging to either anti or syn conformation for adenine and base pairing in all cases. During these simulations, large and dramatic conformational changes are observed that suggest pathways for complex conformational changes in the highly packed DNA interior. Our analysis reveals little difference in the energies of these syn and anti conformations, in contrast to control calculations carried out for standard DNA (in the absence of a neighboring pyrene). While no interconversion between the conformations was observed in standard simulations, reversible anti/syn exchange was directly simulated using the locally enhanced sampling approach. No exchange was seen in the non-pyrene control sequence. Together, these results suggest that an increased flexibility is introduced as a consequence of the pyrene substitution, offering an explanation that is consistent with the available experimental data. These results increase our optimism that simulations in atomic detail may provide accurate models for experimental observations in complex systems.     

Solvent effect on DNA base stacked dimers: An isodensity polarizable continuum model approach

The role of solvent on the base stacking properties of various stacked dimers has been analyzed. Ab initio calculations have been performed on the various stacked dimers using the isodensity polarizable continuum model (IPCM), in the framework of the HF/6‐31G** level. It is observed that the stacked dimers undergo different levels of stabilization for different rotated conformations, and the total energy of stacked dimers depends on the twist angle. The results reveal that DNA stacked dimers prefer the twisted (rotated conformation) conformation in water environment, so as to escape from water, due to their hydrophobic nature. In addition, the presence of solvent stabilizes the stacking interaction between bases. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem, 2001     

The electronic structure evolution of DNA during its conformation transition process

We combined classical molecular dynamics (MD) simulation with ab initio calculations to study the electronic structure evolution of DNA during its conformation transition process. By using MD simulation, we obtained the conformation transition trajectory of an oligonucleotide poly(dC)-poly(dG), from which we selected a series of representative conformations and then performed ab initio calculations for these conformations to reveal their electronic structures. Counterintuitively, the results indicate that during the conformation transition process of DNA, thermal fluctuation plays a more important role than global conformation parameters in affecting the electronic structure of DNA.     

Interpreting NMR data for beta-peptides using molecular dynamics simulations

NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.     

Conformations of poly{G}-poly{C} pi stacks with high hole mobility

Charge transfer properties of DNA depend strongly on the pi stack conformation. In the present paper, we identify conformations of homogeneous poly-{G}-poly-{C} stacks that should exhibit high charge mobility. Two different computational approaches were applied. First, we calculated the electronic coupling squared, V(2), between adjacent base pairs for all 1 ps snapshots extracted from 15 ns molecular dynamics trajectory of the duplex G(15). The average value of the coupling squared is found to be 0.0065 eV(2). Then we analyze the base-pair and step parameters of the configurations in which V(2) is at least an order of magnitude larger than . To obtain more consistent data, approximately 65,000 configurations of the (G:C)(2) stack were built using systematic screening of the step parameters shift, slide, and twist. We show that undertwisted structures (twist<20 degrees) are of special interest, because the pi stack conformations with strong electronic couplings are found for a wide range of slide and shift. Although effective hole transfer can also occur in configurations with twist=30 degrees and 35 degrees, large mutual displacements of neighboring base pairs are required for that. Overtwisted conformation (twist> or =38 degrees) seems to be of limited interest in the context of effective hole transfer. The results may be helpful in the search for DNA based elements for nanoelectronics.     

Molecular Mechanics Investigation of an Adenine-Adenine Non-Canonical Pair Conformational Change

Conformational changes are important in RNA for binding and catalysis and understanding these changes is important for understanding how RNA functions. Computational techniques using all-atom molecular models can be used to characterize conformational changes in RNA. These techniques are applied to an RNA conformational change involving a single base pair within a nine base pair RNA duplex. The Adenine-Adenine (AA) non-canonical pair in the sequence 5'GGUGAAGGCU3' paired with 3'PCCGAAGCCG5', where P is Purine, undergoes conformational exchange between two conformations on the timescale of tens of microseconds, as demonstrated in a previous NMR solution structure [Chen, G., et al., Biochemistry, 2006. 45: 6889-903]. The more populated, major, conformation was estimated to be 0.5 to 1.3 kcal/mol more stable at 30 °C than the less populated, minor, conformation. Both conformations are trans-Hoogsteen/sugar edge pairs, where the interacting edges on the adenines change with the conformational change. Targeted Molecular Dynamics (TMD) and Nudged Elastic Band (NEB) were used to model the pathway between the major and minor conformations using the AMBER software package. The adenines were predicted to change conformation via intermediates in which they are stacked as opposed to hydrogen-bonded. The predicted pathways can be described by an improper dihedral angle reaction coordinate. Umbrella sampling along the reaction coordinate was performed to model the free energy profile for the conformational change using a total of 1800 ns of sampling. Although the barrier height between the major and minor conformations was reasonable, the free energy difference between the major and minor conformations was the opposite of that expected based on the NMR experiments. Variations in the force field applied did not improve the misrepresentation of the free energies of the major and minor conformations. As an alternative, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approximation was applied to predict free energy differences between the two conformations using a total of 800 ns of sampling. MM-PBSA also incorrectly predicted the major conformation to be higher in free energy than the minor conformation.     

Statistics of time-limited ensembles of bent DNA conformations

The paper considers statistical properties of ensembles of chain conformations obtained by short-time Brownian dynamics (BD) of a coarse-grained DNA model in order to find out if the conditions necessary for accurate evaluation of the polymer elasticity are attainable in atom-level molecular dynamics (MD) simulations. To measure the bending persistence length (PL) with a 10% error using data accumulated in a single trajectory of a double helix of 15 base pairs, dynamics should be continued for a few microseconds. However, these estimates should be scaled down by about 2 orders of magnitude because the bending dynamics of short double helices in MD features much smaller relaxation times. As a result, good qualitative agreement with the worm-like chain (WLC) theory is reached in MD after tens of nanoseconds. The presently accessible durations of MD trajectories provide reasonably accurate evaluation of DNA elasticity and allow modeling of its mesoscopic properties. The surprisingly fast bending dynamics of short double helices in MD suggests that the microscopic mechanisms of DNA flexibility differ from a simple harmonic model.     

Solution NMR Structure of a Ligand/Hybrid‐2‐G‐Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding

Telomeric G‐quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G‐quadruplex that adopts the biologically relevant hybrid‐2 conformation in a ligand‐bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G‐quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G‐quadruplex. The ligand is sandwiched between one terminal G‐tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G‐quadruplex structure as observed for other G‐quadruplexes in different conformations, invalidating simple docking approaches to ligand–G‐quadruplex structure determination.     

Insights into the dynamics of HIV-1 protease: a kinetic network model constructed from atomistic simulations

The conformational dynamics in the flaps of HIV-1 protease plays a crucial role in the mechanism of substrate binding. We develop a kinetic network model, constructed from detailed atomistic simulations, to determine the kinetic mechanisms of the conformational transitions in HIV-1 PR. To overcome the time scale limitation of conventional molecular dynamics (MD) simulations, our method combines replica exchange MD with transition path theory (TPT) to study the diversity and temperature dependence of the pathways connecting functionally important states of the protease. At low temperatures the large-scale flap opening is dominated by a small number of paths; at elevated temperatures the transition occurs through many structurally heterogeneous routes. The expanded conformation in the crystal structure 1TW7 is found to closely mimic a key intermediate in the flap-opening pathways at low temperature. We investigated the different transition mechanisms between the semi-open and closed forms. The calculated relaxation times reveal fast semi-open ? closed transitions, and infrequently the flaps fully open. The ligand binding rate predicted from this kinetic model increases by 38-fold from 285 to 309 K, which is in general agreement with experiments. To our knowledge, this is the first application of a network model constructed from atomistic simulations together with TPT to analyze conformational changes between different functional states of a natively folded protein.     

Structure and dynamics of poly(T) single-strand DNA: implications toward CPD formation

The formation of cyclobutane pyrimidine dimers between adjacent thymines by UV radiation is thought to be the first event in a cascade leading to skin cancer. Recent studies showed that thymine dimers are fully formed within 1 ps of UV irradiation, suggesting that the conformation at the moment of excitation is the determining factor in whether a given base pair dimerizes. MD simulations on the 50 ns time scale are used to study the populations of reactive conformers that exist at any given time in T18 single-strand DNA. Trajectory analysis shows that only a small percentage of the conformations fulfill distance and dihedral requirements for thymine dimerization, in line with the experimentally observed quantum yield of 3%. Plots of the pairwise interactions in the structures predict hot spots of DNA damage where dimerization in the ssT18 is predicted to be most favored. The importance of hairpin formation by intra-strand base pairing for distinguishing reactive and unreactive base pairs is discussed in detail. The data presented thus explain the structural origin of the results from the ultrafast studies of thymine dimer formation.     

DNA-mediated charge transport requires conformational motion of the DNA bases: elimination of charge transport in rigid glasses at 77 K

We have proposed that DNA-mediated charge transport (CT) is gated by base motions, with only certain base conformations being CT-active; a CT-active conformation can be described as a domain, a transiently extended pi-orbital defined dynamically by DNA sequence. Here, to explore these CT-active conformations, we examine the yield of base-base CT between photoexcited 2-aminopurine (Ap*) and guanine in DNA in rigid LiCl glasses at 77 K, where conformational rearrangement is effectively eliminated. Duplex DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)nG (n = 0-4). The yield of CT was monitored through fluorescence quenching of Ap* by G. We find, first, that the emission intensity of Ap* in all DNA duplexes increases dramatically upon cooling and becomes comparable to free Ap*. This indicates that all quenching of Ap* in duplex DNA is a dynamic process that requires conformational motion of the DNA bases. Second, DNA-mediated CT between Ap* and G is not observed at 77 K; rather than hindering the ability of DNA to transport charge, conformational motion is required. Moreover, the lack of DNA-mediated CT at 77 K, even through the shortest bridge, suggests that the static structures adopted upon cooling do not represent optimum CT-active conformations. These observations are consistent with our model of conformationally gated CT. Through conformational motion of the DNA bases, CT-active domains form and break-up transiently, both facilitating and limiting CT.     

Preparation and characterization of inclusion complexes of topotecan with sulfonatocalixarene

Inclusion complex of sulfonatocalix[4]arene (SC4A) with topotecan (TPT) was prepared, and its inclusion complexation behaviors, such as stoichiometry, complex stability constants, and inclusion mode, were investigated by means of UV/Vis spectroscopy, DSC, and 2D NMR. The obtained results show that the quinoline ring and the dimethylaminomethyl group of TPT can be efficiently encapsulated in SC4A, and the complex is more soluble than free TPT.     

Vibrational dynamics of DNA: IV. Vibrational spectroscopic characteristics of A-, B-, and Z-form DNA's

Linear and nonlinear IR spectroscopic studies of nucleic acids can provide crucial information on solution conformations of DNA double helix and its complex with other molecules. Carrying out density functional theory calculations of A-, B-, and Z-form DNA's, the authors obtained vibrational spectroscopic properties as well as coupling constants between different basis modes. The vibrational couplings that determine the extent of exciton delocalization are strongly dependent on DNA conformation mainly because the interlayer distance between two neighboring base pairs changes with respect to the DNA conformation. The Z-DNA has comparatively small interlayer vibrational coupling constants so that its vibrational spectrum depends little on the number of base pairs, whereas the A-DNA shows a notable dependency on the size. Furthermore, it is shown that a few distinctively different line shape changes in both IR and two-dimensional IR spectra as the DNA conformation changes from B to A or from B to Z can be used as marker bands and characteristic features distinguishing different DNA conformations.     

Electrospray ionization ion mobility spectrometry of carboxylate anions: ion mobilities and a mass-mobility correlation

A number of carboxylate anions spanning a mass range of 87-253 amu (pyruvate, oxalate, malonate, maleate, succinate, malate, tartarate, glutarate, adipate, phthalate, citrate, gluconate, 1,2,4-benzenetricarboxylate, and 1,2,4,5-benzenetetracarboxylate) were investigated using electrospray ionization ion mobility spectrometry. Measured ion mobilities demonstrated a high correlation between mass and mobility in both N2 and CO2 drift gases. Such a strong mass-mobility correlation among structurally dissimilar ions suggests that the carboxylate functional group that these ions have in common is the source of the correlation. Computational analysis was performed to determine the most stable conformation of the studied carboxylate anions in the gas phase under the current experimental conditions. This analysis indicated that the most stable conformations for multicarboxylate anions included intramolecular hydrogen-bonded ring structures formed between the carboxylate group and the neutral carboxyl group. The carboxylate anions that form ring confirmations generally show higher ion mobility values than those that form extended conformations. This is the first observation of intramolecular hydrogen-bonded ring conformation of carboxylate anions in the gas phase at atmospheric pressure.     

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.     

DFT study of polymorphism of the DNA double helix at the level of dinucleoside monophosphates

We apply DFT calculations to deoxydinucleoside monophosphates (dDMPs) which represent minimal fragments of the DNA chain to study the molecular basis of stability of the DNA duplex, the origin of its polymorphism and conformational heterogeneity. In this work, we continue our previous studies of dDMPs where we detected internal energy minima corresponding to the “classical” B conformation (BI‐form), which is the dominant form in the crystals of oligonucleotide duplexes. We obtained BI local energy minima for all existing base sequences of dDMPs. In the present study, we extend our analysis to other families of DNA conformations, successfully identifying A, BI, and BII energy minima for all dDMP sequences. These conformations demonstrate distinct differences in sugar ring puckering, but similar sequence‐dependent base arrangements. Internal energies of BI and BII conformers are close to each other for nearly all the base sequences. The dGpdG, dTpdG, and dCpdA dDMPs slightly favor the BII conformation, which agrees with these sequences being more frequently experimentally encountered in the BII form. We have found BII‐like structures of dDMPs for the base sequences both existing in crystals in BII conformation and those not yet encountered in crystals till now. On the other hand, we failed to obtain dDMP energy minima corresponding to the Z family of DNA conformations, thus giving us the ground to conclude that these conformations are stabilized in both crystals and solutions by external factors, presumably by interactions with various components of the media. Overall the accumulated computational data demonstrate that the A, BI, and BII families of DNA conformations originate from the corresponding local energy minimum conformations of dDMPs, thus determining structural stability of a single DNA strand during the processes of unwinding and rewinding of DNA. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem 110:2548–2559, 2010     

Electronic Interactions of Michler's Ketone with DNA Bases in Synthetic Hairpins

The mechanism and dynamics of photoinduced electron transfer in two families of DNA hairpins possessing Michler's ketone linkers have been investigated by means of steady state and time‐resolved transient absorption and emission spectroscopies. The excited state behavior of the diol linker employed in hairpin synthesis is similar to that of Michler's ketone in methanol solution. Hairpins possessing only a Michler's ketone linker undergo fast singlet state charge separation and charge recombination with an adjacent purine base, attributed to well‐stacked ground state conformations, and intersystem crossing to the triplet state, attributed to poorly stacked ground state conformations. The failure of the triplet to undergo electron transfer reactions on the 7 ns time scale of our measurements is attributed to the low triplet energy and reduction potential of the twisted triplet state. Hairpins possessing both a Michler's ketone linker and a perylenediimide base surrogate separated by four base pairs undergo photoinduced hole transport from the diimide to Michler's ketone upon excitation of the diimide. The efficiency of hole transport is dependent upon the sequence of the intervening purine bases.