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Hydrogen sulfide, Nitrogen Dioxide, and sulfur dioxide are highly toxic gases with health effects at ppb and ppm levels in air. These compounds occur in industrial operations as well as in environmental atmospheric polluted air. Traditionally, microfabricated electrochemical gas sensors use photolithography processes for patterning of the electrodes and defining the electrode geometry for high surface area to increase the sensor gas sensitivity. We present herein a series of gas sensor designs fabricated via laser ablation of sputtered Au films. For these sensors, the laser ablation step itself takes less than two hours to pattern up to 60 sensors. The sensors are fabricated on a porous hydrophobic substrate made of layers of a Teflon woven mesh, and the 400 nm gold film was sputtered onto a 100 nm tungsten adhesion layer. A total of 60 sensors was patterned in a 6 by 10 grid per substrate with each sensor having a footprint of 15 mm x 15 mm. An image of some of the sensors and their corresponding designs are illustrated in figure 1. Laser ablation was performed by an Optec WS-Flex USP femtosecond 1030 nm laser. The pulse frequency was set to 400 kHz and both the speed and jump speed were set to 200 mm/s. The laser power is nominally 4 W and the power percentage setting was determined for each substrate by patterning a test array onto the substrate in increments of 2%. From this test array, the lowest power that completely ablated the gold could be determined and that was used for the remaining sensor ablation. Images of the etched surface area shown in the figure 1. Surface analysis with EDS using SEM microscope indicated the composition of the layers and effective removal of the gold from the Teflon sheet without damage to the Teflon porosity in the woven mesh, figure 2. The sensor electrodes were assembled by lamination between several plastic layers and then an electrolyte was added. The assembled sensors were diced from the assembled 10x6 sensor wafer and individual sensors were connected to a potentiostat and tested via exposure to the various pollutant gases. The current response of these amperometric sensors was measured and found to be linear with respect to concentration in the low ppm range. This work illustrates an alternative to photolithography for the preparation of thin film gas porous electrodes for use in amperometric gas sensors. Figure 1

Recently, microneedle patches have been explored for extracting interstitial fluid with the goal of extracting temporally relevant, clinical-grade information for human health monitoring. As compared to traditional hypodermic needles, the sub-millimeter scale of microneedles allows for the creation of micropores providing access into human skin interstitial fluid while minimizing interactions with blood vessels and nerves, leading to painless insertion with little to no bleeding. An essential sub-component is the actuator, responsible for driving the microneedle into the skin with a precise force and velocity to ensure reliable insertion. Reliability, in this case, consists of two criteria: the ability of the microneedle to 1) penetrate the skin, and 2) withstand penetration forces without mechanical failure. Evaluation of these criteria requires a thorough understanding of the non-linear, time-dependent interactions between the microneedle and the skin during insertion, including rupture and tearing of the skin on the micron scale, and the resultant stresses on the microneedle. To this end, a comprehensive finite-element model was developed to simulate the microneedle insertion process. This analysis yielded a prediction of complete microneedle insertion without failure of the microneedle and an estimated insertion force of 0.055 N per microneedle, well within the capability of the actuator system considered. This insertion force was validated using experimental data obtained through microneedle insertion in whole skin samples. The model was then used to perform several parametric studies, yielding valuable insights for possible future design improvements.

This paper presents fabrication approaches for magnesium (Mg) microstructures embedded in biodegradable polymers using through-mold Mg electrodeposition and metal- transfer-molding. Biodegradable implantable electronics have garnered increasing interest from the medical community for the monitoring and treatment of transient diseases. Magnesium is a biodegradable metal with desirable properties, and the ability to micropattern Mg thick films (i.e., about >1 µm) with direct microelectromechanical systems (MEMS) integration would sup- port the development of more sophisticated and clinically relevant biodegradable devices and microsystems. Magnesium microstruc- tures were electroplated through micropatterned water-soluble molds in a nonaqueous electrolyte and transfer molded into a biodegradable polymer. Electroplated Mg compared favorably with commercial Mg foil based on elemental composition, crystal orientation, electrical resistivity, and corrosion behavior. Mag- nesium electroplated to a thickness of up to 50 µ ms howed a grain size of ∼10 µm, and minimum feature dimensions of 100 µm in width and spacing. Completely biodegradable Mg and poly-L-lactic acid constructs were demonstrated. The application of Mg thick films toward biodegradable energy sources was explored through the fabrication and testing of biodegradable Mg/Fe batteries. The batteries exhibited a capacity and power of up to 2.85 mAh and 39 µW, respectively. Results confirmed the advantages of electrodeposited Mg microstructures for biodegradable MEMS applications. (2014-0103)

Metallic magnetic materials have desirable magnetic properties, including high permeability, and high saturation flux density, when compared with their ferrite counterparts. However, eddy-current losses preclude their use in many switching converter applications, due to the challenge of simultaneously achieving sufficiently thin laminations such that eddy currents are suppressed (e.g., 500 nm-1 μm for megahertz frequencies), while simultaneously achieving overall core thicknesses such that substantial power can be handled. A CMOS-compatible fabrication process based on robot-assisted sequential electrodeposition followed by selective chemical etching has been developed for the realization of a core of substantial overall thickness (tens to hundreds of micrometers) comprised of multiple, stacked permalloy (Ni80Fe20) nanolaminations. Tests of toroidal inductors with nanolaminated cores showed negligible eddy-current loss relative to total core loss even at a peak flux density of 0.5 T in the megahertz frequency range. To illustrate the use of these cores, a buck power converter topology is implemented with switching frequencies of 1-2 MHz. Power conversion efficiency greater than 85% with peak operating flux density of 0.3-0.5 T in the core and converter output power level exceeding 5 W was achieved.

Bio-electrodes have traditionally been made of materials such as metal and silicon that are much stiffer than the tissue from which they record or stimulate. This difference in mechanical compliance can cause incomplete or ineffective contact with the tissue. The electrode stiffness has also been hypothesized to cause chronic low-grade injury and scar-tissue encapsulation, reducing stimulation and recording efficiency. As an initial step to resolve these issues with electrode performance, we have developed and characterized electrically-functional, low-Young's modulus, microcable-shaped neuroelectrodes and demonstrated electrophysiological recording functionality. The microcable geometry gives the electrodes a similar footprint to traditional wire and microwire neuroelectrodes, while reducing the difference in Young's modulus from nervous tissue by orders of magnitude. The electrodes are composed of PDMS and thin-film gold, affording them a high-level of compliance that is well suited for in vivo applications. The composite Young's modulus of the electrode was experimentally determined to be 1.81 ± 0.01 MPa. By incorporating a high-tear-strength silicone, Sylgard 186, the load at failure was increased by 92%, relative to that of the commonly used Sylgard 184. The microcable electrodes were also electromechanically tested, with measurable conductivity (220 kΩ) at an average 8% strain (n = 2) after the application of 200% strain. Electrophysiological recording is demonstrated by wrapping the electrode around a peripheral nerve, utilizing the compliance and string-like profile of the electrode for effective recording in nerve tissue.

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G2T2G2TGTG2T2G2), PS2.M, a catalytically active aptamer (d(GTG3TAG3CG3T2G2)), and the human telomere repeat (HT) (d(AG3(T2AG3)3)). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 302–309, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The conformation of the guanine quadruplex formed by the human telomere (HT) repeat in solutions containing physiological concentrations of K+ ions has been a topic of intensive investigation during the past several years. Of particular interest are the directionality of the overall folding pattern, i.e., parallel, antiparallel, or a combination of these two modes, and the alternation, if any, of the glycosidic bond conformation between syn and anti. An additional issue involves resolving mixtures of conformations when more than one species is present. We approach these questions using selective substitution of riboguanosine, rG, for deoxyriboguanosine, dG. Using a combination of circular dichroism, gel electrophoresis, equilibrium ultracentrifugation, and imino proton NMR, we are able to show that these modifications can yield sequences which fold into parallel or antiparallel conformations consisting of one or two strands. We also demonstrate that chimeric editing of the HT sequence permits isolating one of two conformational isomers existing in solution in the presence of KCl. The ability to engineer and control quadruplex folding motifs illustrated here with HT may prove useful more generally for a variety of quadruplex-forming sequences.

Planar or two-dimensional (2D) microelectrode arrays (MEAs), which are used for in vitro culturing of neurons and tissue slices, have been in existence for over 30 years. However, in order to study complex network morphologies and tissue slices which contain substantial 3D neuronal structures, 3D MEAs with microfluidic ports are required. Integrated fabrication of 3D MEAs with embedded microfluidic ports for nutrient perfusion through these relatively thick tissues typically requires non-planar lithography, which is not easily accomplished. This paper reports a laser-scribing technique coupled with electroplating to fabricate 3D MEAs coupled with microfluidic ports. An excimer laser has been used to define patterns in a polymer mold layer that is conformally vapor-deposited on a 3D microfluidic SU-8 substrate. Metal is electroplated through this mold to fabricate electrodes at multiple heights. To demonstrate 3D MEAs, a standard design was chosen consisting of an array of three-dimensional protrusions (‘towers’) optionally with microfluidic functionality on which electrodes can be formed extending to the top of each tower. Additional electrodes are formed on the substrate resulting in a multi-level electrode structure. Since microfluidics can exist both in the substrate as well as along the towers, a coupled three-dimensional electrical and microfluidic functionality is achieved. The resulting 3D MEAs have been analyzed electrically using impedance spectroscopy and baseline noise measurements. They have further been evaluated fluidically using micro-particle image velocimetry measurements.

Nucleic acid quadruplexes, based on the guanine quartet, can arise from one or several strands, depending on the sequence. Those consisting of a single strand are usually folded in one of two principal topologies: antiparallel, in which all or half of the guanine stretches are antiparallel to each other, or parallel, in which all guanine stretches are parallel to each other. In the latter, all guanine nucleosides possess the anti conformation about the glycosidic bond, while in the former, half possess the anti conformation, and half possess the syn conformation. While antiparallel is the more common fold, examples of biologically important, parallel quadruplexes are becoming increasingly common. Thus, it is of interest to understand the forces that determine the quadruplex fold. Here, we examine the influence of individual nucleoside conformation on the overall folding topology by selective substitution of rG for dG. We can reverse the antiparallel fold of the thrombin binding aptamer (TBA) by this approach. Additionally, this substitution converts a unimolecular quadruplex into a bimolecular one. Similar reverse substitutions in the all-RNA analogue of TBA result in a parallel to antiparallel change in topology and alter the strand configuration from bimolecular to unimolecular. On the basis of the specific substitutions made, we conclude that the strong preference of guanine ribonucleosides for the anti conformation is the driving force for the change in topology. These results demonstrate how conformational properties of guanine nucleosides govern not only the quadruplex folding topology but also impact quadruplex molecularity and provide a means to control these properties.

Stabilization of nucleic acid structures results from a balance of multiple interactions, including electrostatics, base stacking, hydrophobic interactions, hydrogen bonding, van der Waals forces, etc. Nucleic acid quadruplexes are unusual structures in that their formation is driven by specific binding of metal ions. This unique mode of metal binding, which is tightly coupled to oligonucleotide folding, can engender correspondingly unique solution behavior. In particular, we show that addition of many cosolvents, such as primary aliphatic alcohols, increases the thermal stability of quadruplexes, as determined by melting temperature, Tm, in direct contrast to the response of duplexes to the same admixture of solvents. Thermal stability is observed to increase as the dielectric constant of the composite solvent decreases. This behavior suggests a dominant role for electrostatics in quadruplex formation and stability. Additional studies done with other cosolvents and solutes suggest that, in some cases, other forces may come into play, including the possibility of direct interaction with the quadruplex structure. Nonetheless, many cosolvents and small molecules, such as ethanol, dimethylformamide, and betaine, stabilize the quadruplex conformation in sharp distinction to their destabilization of DNA duplexes. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 91–101, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Recent X-ray crystallographic studies on the human telomere sequence d[AGGG(TTAGGG)3] revealed a unimolecular, parallel quadruplex structure in the presence of potassium ions, while earlier NMR results in the presence of sodium ions indicated a unimolecular, antiparallel quadruplex. In an effort to identify and isolate the parallel form in solution, we have successfully ligated into circular products the single-stranded human telomere and several modified human telomere sequences in potassium-containing solutions. Using these sequences with one or two terminal phosphates, we have made chemically ligated products via creation of an additional loop. Circular products have been identified by polyacrylamide gel electrophoresis, enzymatic digestion with exonuclease VII and electrospray mass spectrometry in negative ion mode. Optimum pH for the ligation reaction of the human telomere sequence ranges from 4.5 to 6.0. Several buffers were also examined, with MES yielding the greatest ligation efficiency. Human telomere sequences with two phosphate groups, one each at the 3′ and 5′ ends, were more efficient at ligation, via pyrophosphate bond formation, than the corresponding sequences with only one phosphate group, at the 5′ end. Circular dichroism spectra showed that the ligation product was derived from an antiparallel, single-stranded guanine quadruplex rather than a parallel single-stranded guanine quadruplex structure.

Nucleic acid quadruplexes are composed of guanine quartets stabilized by specific metal ions. X-ray diffraction can provide high-resolution information on the structure and metal binding properties of quadruplexes, but only if they can be crystallized. NMR can provide detailed information on the solution structure of such quadruplexes but little quantitative data concerning the metal binding site. Here we apply extended X-ray absorption fine structure (EXAFS) measurements to characterize the metal ion binding site, in frozen solution, of the unimolecular quadruplex formed by the thrombin binding aptamer, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)) (TBA), in the presence of Pb(2+) ions. The Pb L(III) -edge X-ray absorption spectrum of this metal-DNA complex is very similar to that we obtain for a Pb(2+)-stabilized quartet system of known structure constructed from a modified guanine nucleoside (G1). The Fourier transforms of the Pb(2+) complexes with both TBA and G1 show a first-shell interaction at about 2.6 A, and a weaker, broader shell at 3.5-4.0 A. Quantitative analysis of the EXAFS data reveals the following: (i) very close agreement between interatomic distances at the metal coordination site for the Pb(2+)-G1 complex determined by EXAFS and by X-ray crystallography; (ii) similarly close agreement between interatomic distances measured by EXAFS for the Pb(2+)-G1 and Pb(2+)-TBA complexes. These results provide strong evidence for binding of the Pb(2+) ion in the region between the two quartets in the Pb(2+)-TBA complex, coordinated to the eight surrounding guanine O6 atoms. The specific binding of Pb(2+) to DNA examined here may be relevant to the genotoxic effects of this environmentally important heavy metal. Furthermore, these results demonstrate the utility of EXAFS as a method for quantitative characterization of specific metal binding sites in nucleic acids in solution.

The thrombin aptamer is a 15-mer oligodeoxyribonucleotide that folds into a unimolecular quadruplex consisting of a stack of two guanine quartets connected by two external loops and one central loop and possesses a high affinity for thrombin. We have undertaken a systematic examination, in KCl, of the thermodynamic stability of thrombin aptamer analogues containing sequence modifications in one or more of the loops, as well as in the number of quartets. UV melting studies have been carried out to obtain the relevant thermodynamic parameters for these aptamers. van't Hoff analysis of these data, with a two-state model for unimolecular denaturation, gave excellent fits to the experimental observations. Thermodynamic analysis indicates that the central loop sequence in the parent aptamer is optimal for stability. Modifications in this or other loops can effect either DeltaH degrees, DeltaS degrees, or both. Addition of a single G at the 5'-end decreases stability while addition of a G at the 3'-end increases stability. Differential scanning calorimetry experiments on the thrombin aptamer reveal that a heat capacity change, not detected by UV measurements, accompanies the unfolding of the aptamer.

Among the many unusual conformations of DNA and RNA, quadruplex structures, based on the guanine quartet, possess several unique properties. These properties, along with the general features of guanine quadruplexes, are described in the context of possible roles for these structures in biological systems. A variety of experimental observations supporting the notion that quadruplexes are important in vivo is presented, including proteins known to specifically bind to quadruplex structures, guanine-rich DNA, and RNA sequences endowed with the potential for forming quartet-based structures in telomeres and regulatory regions, such as gene promoters, quadruplexes as DNA aptamer folding motifs arising from in vitro selection experiments, and potential chemotherapeutic, quadruplex-forming oligonucleotides. Taken together, all of these observations argue cogently not only for the presence of quadruplexes in biological systems but also for their significance in terms of their roles in various biological processes. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 56: 209–227, 2001

The solution structure and dynamical properties of the potassium-stabilized, hairpin dimer quadruplex formed by the oligonucleotide d(G3T4G3) have been elucidated by a combination of high-resolution NMR and molecular dynamics simulations. Refinement calculations were carried out both in vacuo, without internally coordinated K+ cations, and in explicit water, with internally coordinated K+ cations. In the latter case, the electrostatic interactions were calculated using the particle mesh Ewald (PME) method. The NMR restraints indicate that the K+ quadruplex has a folding arrangement similar to that formed by the same oligonucleotide in the presence of sodium, but with significant local differences. Unlike the Na+ quadruplex, the thymine loops found in K+ exhibit considerable flexibility, and appear to interconvert between two preferred conformations. Furthermore, the NMR evidence points toward K+-stabilized guanine quartets of slightly larger diameter relative to the Na+-stabilized structure. The characteristics of the quartet stem are greatly affected by the modeling technique employed: caged cations alter the size and symmetry of the quartets, and explicit water molecules form hydration spines within the grooves. These results provide insight into those factors that determine the overall stability of hairpin dimer quadruplexes and the effects of different cations in modulating the relative stability of the dimeric hairpin and linear, four-stranded, quadruplex forms.

Ligands capable of specific recognition of RNA structures are of interest in terms of the principles of molecular recognition as well as potential chemotherapeutic applications. We have approached the problem of identifying small molecules with binding specificity for the RNA double helix through application of the DOCK program [Kuntz, I. D., Meng, E. C., and Shoichet, B. K. (1994) Acc. Chem. Res. 27, 117-123], a structure-based method for drug discovery. A series of lead compounds was generated through a database search for ligands with shape complementarity to the RNA deep major groove. Compounds were then evaluated with regard to their fit into the minor groove of B DNA. Those compounds predicted to have an optimal fit to the RNA groove and strong discrimination against DNA were examined experimentally. Of the 11 compounds tested, 3, all aminoglycosides, exhibited pronounced stabilization of RNA duplexes against thermal denaturation with only marginal effects on DNA duplexes. One compound, lividomycin, was examined further, and shown to facilitate the ethanol-induced B to A transition in calf thymus DNA. Fluorine NMR solvent isotope shift measurements on RNA duplexes containing 5-fluorouracil provided evidence that lividomycin binds in the RNA major groove. Taken together, these results indicate that lividomycin recognizes the general features of the A conformation of nucleic acids through deep groove binding, confirming the predictions of our DOCK analysis. This approach may be of general utility for identifying ligands possessing specificity for additional RNA structures as well as other nucleic acid structural motifs.

We have targeted the d[G(AG)5]. d[C(TC)5] duplex for triplex formation at neutral pH with either d[G(AG)5] or d[G(TG)5]. Using a combination of gel electrophoresis, uv and CD spectra, mixing and melting curves, along with DNase I digestion studies, we have investigated the stability of the 2:1 pur*pur.pyr triplex, d[G(AG)5]*d[G(AG)5].d[C(TC)5], in the presence of MgCl2. This triplex melts in a monophasic fashion at the same temperature as the underlying duplex. Although the uv spectrum changes little upon binding of the second purine strand, the CD spectrum shows significant changes in the wavelength range 200-230 nm and about a 7 nm shift in the positive band near 270 nm. In contrast, the 1:1:1 pur/pyr*pur.pyr triplex, d[G(TG)5]*d[G(AG)5].d[C(TC)5], is considerably less stable thermally, melting at a much lower temperature than the underlying duplex, and possesses a CD spectrum that is entirely negative from 200 to 300 nm. Ethidium bromide undergoes a strong fluorescence enhancement upon binding to each of these triplexes, and significantly stabilizes the pur/pyr*pur.pyr triplex. The uv melting and differential scanning calorimetry analysis of the alternating sequence duplex and pur*pur.pyr triplex shows that they are lower in thermodynamic stability than the corresponding 10-mer d(G3A4G3). d(C3T4C3) duplex and its pur*pur.pyr triplex under identical solution conditions.

The mechanical and electrochemical properties of magnesium are favorable for biomedical and energy storage applications. However, magnesium microfabrication has been limited to sub-micron-thick film technologies (i.e., sputtering or evaporation). This paper presents three magnesium microfabrication approaches for thicknesses greater than 10 μm: 1) laser-cutting and 2) chemical etching of 70-μm-thick commercial magnesium foil; and 3) through-mold electroplating of magnesium from non-aqueous solution. The fabrication technologies are compared on minimum feature size, morphology, uniformity, composition and electrical resistivity. Preliminary results confirmed that the 50-μm-thick electroplated material composition compared favorably with commercial magnesium foil. The measured electrical resistivities of commercial and electrodeposited magnesium were 5.3 μΩ·cm and 8.7 μΩ·cm, respectively. Thick magnesium microstructures can be fabricated through several means to serve a broad range of MEMS-based applications.

Two-dimensional 1H NMR studies on the dimeric hairpin quadruplex formed by d(G3T4G3) in the presence of either NaCl or KCl are presented. In the presence of either salt, the quadruplex structure is characterized by half the guanine nucleosides in the syn conformation about the glycosidic bond, the other half in the anti conformation, as reported for other similar sequences. However, 1H NOESY and 1H-31P heteronuclear correlation experiments demonstrate that the deoxyguanosines do not strictly alternate between syn and anti along individual strands. Thus we find the following sequences with regard to glycosidic bond conformation: 5'-G1SG2SG3AT4AT5A-T6AT7AG8SG9AG10A-3' and 5'-G11SG12AG13AT14AT1 5AT16AT17AG18SG19SG20A-3', where S and A denote syn and anti, respectively. This represents the first experimental evidence for a nucleic acid structure containing two sequential nucleosides in the syn conformation. The stacking interactions of the resulting quadruplex quartets and their component bases have been evaluated using unrestrained molecular dynamics calculations and energy component analysis. These calculations suggest that the sequential syn-syn/anti-anti and syn-anti quartet stacks are almost equal in energy, whereas the anti-syn stack, which is not present in our structure, is energetically less favorable by about 4 kcal/mol. Possible reasons for this energy difference and its implications for the stability of quadruplex structures are discussed.

Interaction of the antimalarial drugs quinacrine and chloroquine with DNA has been studied extensively in order to understand the origin of their biological activity. These studies have shown that they bind to DNA through an intercalative mode and show little sequence specificity. All previous experiments were carried out using the racemic form of these drugs. We have investigated the binding of the enantiomeric forms of quinacrine and chloroquine to synthetic polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly (dG-dC), and found interesting differences in their binding parameters. Quinacrine enantiomers have a much higher binding affinity for the two polynucleotides compared to those of chloroquine. The negative enantiomers were found to have higher binding affinity than the positive ones. The binding constant for the binding of quinacrine(-) to poly(dG-dC).poly(dG-dC) was found to be about 3 times that of quinacrine(+). The differences in these binding affinities were further confirmed by equilibrium dialysis of the complexes of the polynucleotides with the racemic form of the drugs, which resulted in the enrichment of the dialysate with the positive enantiomer. CD spectra of the enantiomers and their polynucleotide complexes are reported. Changes in the fluorescence properties of quinacrine in the presence of the two polynucleotides are also described. Biological implications of these findings are discussed.

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

In an effort to probe the structure and stability of the CH + -C base pair in DNA, we have studied the conformation of the deoxydecanucleotide d(C 3 T 4 C 3 ) at pH 5.5, using 1 H-NMR and UV absorption spectroscopy. Lowering the pH of a solution containing d(C 3 T 4 C 3 ) is accompanied by the induction of six new imino proton NMR resonances in a region (15.415.8 ppm) downfield from where either T-H 3 or G-H1 Watson-Crick (W-C)-paired imino protons normally resonate

Two-dimensional (2D) 1H-NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self-complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B-type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A.T-base-pair region. At 18 degrees C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil-d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry-related binding sites, which span the 5'-TAA-3' region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil-d(GCAATTGC)2 complex.

The binding of spermine to the d(m 5 CGm 5 CGm 5 CG) duplex has been studied by proton and phosphorus nuclear magnetic resonance techniques in order to investigate the mobility and nature of spermine bound to the resulting Z -DNA complex. A characterization of the B to Z transition as a function of increasing spermine concentration demonstrated doubling of the non-exchangeable proton and the phosphorus peaks at a ratio of about 1:1 (spermine/duplex) and a re-simplification of the spectrum at 2:1 (spermine/duplex) where about 90% or the DNA was fully converted into the Z -form. However, some of the Z -DNA proton chemical shifts differed between the 1:1 and 2:1 titration points. Since these differences involved primarily the exchangeable terminal imino and amino protons, they could result from end effects. Discrepancies were generally not observed with the non-terminal proton shifts nor with the phosphorus shifts. These proton and phosphorus chemical shift changes are fully consistent with a, B to Z transition. Complexed spermine peaks appear about 0.1 parts per million upfield from the uncomplexed form. The spermine and both the B and Z -DNA hexamer signals are noticeably broadened at the 1:1 ratio but the remaining signals re-sharpen at the 2:1 ratio. Both one-dimensional and two-dimensional studies revealed negative nuclear Overhauser effect (NOE) contacts between each spermine proton. Therefore, spermine has a longer correlation time than that observed for unbounded spermine. These results are contrasted with the positive NOE contacts observed for the B -DNA-spermine complexes reported by Wemmer et al. using the dodecamer d(CGCGAATTCGCG) 2 and reported here using the hexamer d(ATGCAT) 2 . While the mobility of spermine in the Z -DNA complex is significantly less than that of the B -DNA complex, no clear evidence of intermolecular spermine-DNA proton NOE contacts is observed.

The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.

In this paper, we report the results of a rapid and room temperature integration approach for the selective and structured deposition of carbon nanotubes (CNTs) into three-dimensional microstructures. The approach exploits electrophoretic deposition (EPD) from an aqueous suspension of CNTs, together with suitably patterned and electrically-energized microstructure-bearing substrates. Uniform 2-D and 3-D micropatterns of CNTs on wafer scale have been achieved in less than 4 minutes with controllable thicknesses ranging from 133nm to several micrometers. Orientation of the deposited CNTs was observed in microstructures with certain dimensions. Surface hydrophobicity of the microstructures was found to be critical in achieving well-defined micropatterning of CNTs. A hydrophobic microstructure surface leads to the selective patterning profiles of CNTs, while a hydrophilic surface induces CNTs assembly over the entire microstructure, with resultant loss of selectivity. This approach can be further extended to fabricate 3-D micropatterns with multilayer materials on flexible substrate through the aid of transfer micromolding techniques.

The binding of mithramycin A to the d(A1T2G3C4A5T6) duplex was investigated by 1H NMR and found to be similar to that of its analogue chromomycin A3. In the presence of Mg2+, mithramycin binds strongly to d(ATGCAT)2. On the basis of the two-dimensional NOESY spectrum, the complex formed possesses C2 symmetry at a stoichiometry of two drugs per duplex (2:1) and is in slow chemical exchange on the NMR time scale. NOESY experiments reveal contacts from the E-pyranose of mithramycin to the terminal and nonterminal adenine H2 proton of DNA and from the drug hydroxyl proton to both G3NH2 protons, C4H1' proton, and A5H1' proton. These data place the drug chromophore and E pyranose on the minor groove side of d(ATGCAT)2. NOE contacts from the A-, B-, C-, and D-pyranoses of mithramycin to several deoxyribose protons suggest that the A- and B-rings are oriented along the sugar-phosphate backbone of G3-C4, while the C- and D-rings are located along the sugar-phosphate backbone of A5-T6. These drug-DNA contacts are very similar to those found for chromomycin binding to d(ATGCAT)2. Unlike chromomycin, the NOESY spectrum of mithramycin at the molar ratio of one drug per duplex reveals several chemical exchange cross-peaks corresponding to the drug-free and drug-bound proton resonances. From the intensity of these cross-peaks and the corresponding diagonal peaks, the off-rate constant was estimated to be 0.4 s-1. These data suggest that the exchange rate of mithramycin binding to d(ATGCAT)2 is faster than that of chromomycin.

The existence of DNA triple helices in vitro has been known for some time. Recent evidence suggesting that DNA triplexes exist in vivo and showing their potential for chemotherapeutic applications has renewed interest in these triple-strand conformations. However, little structural information is currently known about these unusual nucleic acid forms. We have induced and stabilized triple-helical (dA)10.2(dT)10 with MgCl2 at neutral pH. UV mixing curves demonstrate a 1:2 (dA)10 to (dT)10 stoichiometry at suitable MgCl2 concentrations. Thermal denaturation profiles establish a melting mechanism characterized by the initial loss of the third strand, followed by dissociation of the remaining duplex. The circular dichroic spectrum of the triplex form is distinct from that of a duplex equimolar in (dA)10. NMR studies show that magnesium-induced triplex formation is accompanied by an upfield shift of several imino proton resonances present before stabilization of the triplex form with MgCl2 and the induction of new upfield imino proton resonances. Nuclear Overhauser effect spectroscopy measurements on both undeuterated and C8--H-deuterated (dA)10.2(dT)10 triplexes demonstrate dipolar contacts between resolvable imino proteins and both adenine C8--H and C2--H aromatic protons. Hence, MgCl2 stabilizes a triplex structure in which thymine N3--H imino protons are involved in both Watson-Crick and Hoogsteen base pairing.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.

This paper reports a laser-assisted fabrication scheme for three-dimensional (3-D) electrodeposited metal structures into high aspect ratio trenches. A polymer (parylene C) is conformally deposited onto a highly nonplanar surface, selectively laser ablated, and used as an electroplating mold to fabricate metallic structures. Laser ablation of high-resolution (< 10 mum) features is performed using a 248 nm KrF Excimer laser. The ablation parameters for the parylene layer have been characterized for various film thicknesses. Metal lines of 2 mum thickness, and 2.5 mum width have been electroplated into 300 mum deep silicon trenches. The minimum resolution achieved is less than 5 mum. This process can potentially be applied toward the fabrication of embedded inductors, high density electrodes, buried interconnects or high voltage circuitry in CMOS and MEMS devices.

This paper reports the design, fabrication and experimental characterization of ultraminiaturized, axial-flux, permanent-magnet (PM) generators intended for milliwatt range systems. The miniaturization of PM machines is attractive because scaling laws are favorable, but integration of ultra-small permanent magnets is challenging. The generators are 2-pole, multi-turn, 2-mm diameter machines. A single-phase, open-circuit voltage of 51 mVrms has been measured at 392 krpm, which approximately corresponds to 3.6 mW of DC power to a load resistance at the point of maximum power transfer, indicating that this approach shows great promises for milliwatt-scale power devices.

In the presence of certain metal ions, DNA and RNA can form guanine quadruplex structures, which have been proposed to play a functional role in a variety of biological processes. An 18-nucleotide DNA oligomer, PS2.M, d(GTG3TAG3CG3T2G2), was previously reported to bind hemin and the resulting complex exhibited peroxidase activity. It was proposed that PS2.M folds unimolecularly into an antiparallel quadruplex with unusual, single-base loops and terminal guanines positioned in adjacent quartets. Here we describe structural and stability properties of PS2.M alone in different buffers and metal ions, using gel electrophoresis, circular dichroism (CD), ultraviolet (UV)-visible spectroscopies, and one-dimensional 1H nuclear magnetic resonance (NMR). Native gel behavior of PS2.M in the presence of either Na+ or Pb2+ suggests the formation of unimolecular structures but, in the presence of K+, both unimolecular and multistranded structures are observed. In the presence of Pb2+ ions, PS2.M forms a unimolecular quadruplex containing three guanine quartets. CD titrations reveal that binding of Pb2+ ions to PS2.M is stoichiometric, and a single lead cation suffices to fully fold PS2.M. The PS2.M–Na+ system also forms a similar unimolecular quadruplex. In the presence of K+, the PS2.M–K+ system forms mixed species. With increasing time and PS2.M concentration, the contribution of unimolecular species decreases while that of multimolecular species increases, and this behavior is independent of buffer media. These results suggest that the catalytically active form, studied in the presence of K+, may be a parallel, multistranded quadruplex rather than an antiparallel, unimolecular quadruplex. © 2006 Wiley Periodicals, Inc. Biopolymers 82:558–569, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

This paper presents a simplified fabrication method to make highly elevated electrical signal lines up to 700 /spl mu/m in height from the substrates. The immediate application is a bioMEMS device that requires high aspect ratio three-dimensional (3D) multi-electrode arrays (MEA) to detect signals from deep within 3D networks of neurons. The demonstrated method generated thick electroplated metal lines on excimer laser patterned parylene molds. The directionality of the laser beam makes it possible to achieve practically straight patterns on the inclined sidewalls in addition to the bottom surface, which are several hundred microns below. 3D multi-electrodes on various elevation surfaces and insulation of parylene coating were efficiently implemented by using the directionality of excimer lasers and the conformal coating of parylene.

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.

The calculation of ligand-nucleic acid binding free energies is investigated by including solvation effects computed with the generalized-Born model. Modifications of the solvation module in DOCK, including introduction of all-atom parameters and revision of coefficients in front of different terms, are shown to improve calculations involving nucleic acids. This computing scheme is capable of calculating binding energies, with reasonable accuracy, for a wide variety of DNA-ligand complexes, RNA-ligand complexes, and even for the formation of double-stranded DNA. This implementation of GB/SA is also shown to be capable of discriminating strong ligands from poor ligands for a series of RNA aptamers without sacrificing the high efficiency of the previous implementation. These results validate this approach to screening large databases against nucleic acid targets. © 2003 Wiley Periodicals, Inc. Biopolymers 73:192–204, 2004

The binding of mithramcyin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.

The loop of four thymines in the sodium form of the dimeric folded quadruplex [d(G3T4G3)]2 assumes a well-defined structure in which hydrogen bonding between the thymine bases appears to contribute to the stability and final conformation of the quadruplex. We have investigated the importance of the loop interactions by systematically replacing each thymine in the loop with a cytosine. The quadruplexes formed by d(G3CT3G3), d(G3TCT2G3), d(G3T2CTG3) and d(G3T3CG3) in the presence of 150 mM Na+ were studied by gel mobility, circular dichroism and 1H NMR spectroscopy. The major species formed by d(G3CT3G3), d(G3TCT2G3) and d(G3T3CG3) at 1 mM strand concentration at neutral pH is a dimeric folded quadruplex. d(G3T2CTG3) has anomalous behaviour and associates into a greater percentage of linear four-stranded quadruplex than the other three oligonucleotides at neutral pH and at the same concentration. The linear four-stranded quadruplex has a greater tendency to oligomerize to larger ill-defined structures, as demonstrated by broad 1H NMR resonances. At pH 4, when the cytosine is protonated, there is a greater tendency for each of the oligonucleotides to form some four-stranded linear quadruplex, except for d(G3T2CTG3), which has the reverse tendency. The experimental results are discussed in terms of hydrogen bonding within the thymine loop.

This review focuses on the structural and thermodynamic characterization of model DNA triplex and quadruplex structures, taking into account effects of stoichiometry and sequence. Methods such as gel electrophoresis, UV melting, and scanning calorimetry, and the results thereof, are described for determination of the thermodynamic stability of such systems. Three classes of triplexes are considered based on the composition of the third strand, while quadruplex systems are limited to those based on the guanine quartet. X-ray crystallography and high resolution NMR studies are also described for these two classes of unusual structures. Ligand binding to triplexes and quadruplexes is also reviewed, with emphasis on specific molecular recognition. The availability of three-dimensional structures for triplex and quadruplex species sets the stage for structure-based development of ligands capable of binding to them specifically. To this end, we consider the application of DOCK, a program for the discovery of small molecules that can recognize macromolecular structures, to the problem of recognizing folded quadruplex structures. Such studies may ultimately lead to pharmaceutically active compounds.

This paper reports three-dimensional (3-D) microfabricated toroidal inductors intended for power electronics applications. A key fabrication advance is the exploitation of thick metal encapsulation of polymer pillars to form a vertical via interconnections. The radial conductors of the toroidal inductor are formed by conventional plating-through-mold techniques, while the vertical windings (up to 650 µm in height) are formed by polymer cores with metal plated on their external surfaces. This encapsulated polymer approach not only significantly reduces the required plating time but also exploits the relative ease of fabricating high-aspect-ratio SU-8 pillars. To form the top radial conductors, non-photopatternable SU-8 is introduced as a thick sacrificial layer. Two toroidal inductor geometries were fabricated and tested. The first inductor had an inner diameter of 2 mm, an outer diameter of 6 mm, 25 turns and a vertical via height of 650 µm. The second inductor had an inner diameter of 4 mm, an outer diameter of 8 mm, 50 turns and a vertical via height of 650 µm. Both inductor geometries were successfully fabricated and characterized in the frequency range of 0.1−100 MHz. Characterization results of the 25- and 50-turn inductors showed an average inductance of 76 and 200 nH, a low frequency (0.1 MHz) resistance of 0.2 and 1 Ω and a quality factor of 35 and 24 at 100 MHz, respectively. Finite-element simulations of the inductors were performed and agreed with the measured results to within 8%. The turn-to-turn breakdown voltage was measured to be in excess of 800 V and currents as high as 0.5 A could be successfully carried by the inductor windings.

We present a thermodynamic analysis based on differential scanning calorimetry (DSC) of three short intermolecular DNA triplexes targeted to the same DNA duplex: d(C+3T4C+3)*d- (G3A4G3).d(C3T4C3) (PYR), d(G3A4G3)*d(G3A4G3).d(C3T4C3) (PUR), and d(G3T4G3)*d(G3A4G3).d(C3T4C3) (PUR/PYR). Enthalpies, delta H, and entropies, delta S, are measured by model-free integration of the DSC curves and are compared to the same quantities determined by van't Hoff analysis of the DSC curves and, in the case of the PYR and PUR/PYR triplexes, UV melting curves as well. In the case of the PUR triplex, which exhibits monophasic melting behavior, the calorimetric delta H and the calorimetrically determined van't Hoff delta H are in excellent agreement, indicating an all-or-none transition for this triplex. For the PYR and PUR/PYR triplexes, which melt in a biphasic manner, the calorimetrically determined van't Hoff delta H values are somewhat larger than the model-independent calorimetric delta H values. In those cases, however, good agreement is found between the calorimetric delta H values and the spectrophotometrically determined van't Hoff delta H values. The calorimetrically determined delta H values, expressed per mole of triplet, for the three triplexes are 4.5, 3.8, and 2.4 kcal/mol for the PUR, PYR, and PUR/PYR triplexes, respectively. The same order of stability is observed in terms of delta G and Tm values. The high stability of the PUR triplex at neutral pH indicates that purine oligonucleotides may be the most effective at targeting duplex regions for triple helix formation in vivo.

The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.

The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G3T4G3), in NaCl solution has been determined by 1H two-dimensional NMR techniques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incorporates the sequences 5'-G1sG2AG3AT4AT5AT6AT7AG8sG9AG10A-3' and 5'-G11sG12AG13AT14AT15AT16AT17AG18sG19sG20A-3' (where S and A denote syn and anti, respectively) in a three-quartet, diagonal-looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447-5455] and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Natl. Acad. Sci. USA 91, 10546-10550] have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals' interactions. The first and third loop thymines stack over the outermost G-quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals' interactions. The unexpected sequential syn-syn deoxyguanosines in the quadruplex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.

We have targeted the d(G3A4G3).d(C3T4C3) duplex for triplex formation with d(G3T4G3) in the presence of MgCl2. The resulting triple helix, d(G3T4G3)*d(G3-A4G3).d(C3T4C3), is considerably weaker than the related triplex, d(G3A4G3)*d(G3A4G3).d(C3T4C3), and melts in a biphasic manner, with the third strand dissociating at temperatures about 20-30 degrees C below that of the remaining duplex. This is in distinct contrast to the d(G3A4G3)*d(G3A4G3).d(C3T4C3) triplex, which melts in essentially a single transition. Gel electrophoresis under non-denaturing conditions shows the presence of the d(G3T4G3)*d(G3A4G3).d(C3T4C3) triplex as a band of low mobility compared to the duplex or the single strand bands. Binding of the d(G3T4G3) third strand and the purine strand of the duplex can be monitored by imino proton NMR spectra. While these spectra are typically very broad for intermolecular triplexes, the line widths can be dramatically narrowed by the addition of two thymines to both ends of the pyrimidine strand. Thermodynamic analysis of UV melting curves shows that this triplex is considerably less stable than related triplexes formed with the same duplex. The orientation of the third strand was addressed by a combination of fluorescence energy transfer and UV melting experiments. Results from these experiments suggest that, in the unlabeled triplex, the preferred orientation of the third strand is parallel to the purine strand of the duplex.

The solution structure of the DNA decamer d(CATTTGCATC)-d(GATGCAAATG), comprising the octamer motif of immunoglobulin genes, is determined by restrained molecular dynamics (rMD) simulations. The restraint data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were previously obtained by a complete relaxation matrix analysis of the two-dimensional nuclear Overhauser enhancement (2D NOE) intensities and by the quantitative simulation of cross-peaks in double-quantum-filtered correlated (2QF-COSY) spectra. The influence of torsion angles and the number of experimental distance restraints on the structural refinement has been systematically examined. Omitting part of the experimental NOE-derived distances results in reduced restraint violations and lower R factors but impairs structural convergence in the rMD refinement. Eight separate restrained molecular dynamics simulations were carried out for 20 ps each, starting from either energy-minimized A- or B-DNA. Mutual atomic root-mean-square (rms) differences among the refined structures are well below 1 A and comparable to the rms fluctuations of the atoms about their average position, indicating convergence to essentially identical structures. The average refined structure was subjected to an additional 100 ps of rMD simulations and analyzed in terms of average torsion angles and helical parameters. The B-type duplex exhibits clear sequence-dependent variations in its geometry with a narrow minor groove at the T3.A3 tract and a large positive roll at the subsequent TG.CA step. This is accompanied by a noticeable bend of the global helix axis into the major groove. There is also evidence of significant flexibility of the sugar-phosphate backbone with rapid interconversion among different conformers.

The ends of chromosomes contain repeats of guanine-rich sequences that can assume highly compact conformations and are presumed necessary for their biological role in chromosomal stabilization and association. We have investigated the conformational behavior of d(G3T4G3) as a function of the addition of either KCl or NaCl, in the concentration range of 50-200 mM, by using a spectrum of physical techniques and conclude that these salts induce a quadruplex species composed of two strands, each in a hairpin conformation. When salt is added, a large positive signal appears near 290 nm in the CD spectra. UV thermal denaturation curves show a single concentration-dependent transition and provide data for quantitating the thermodynamics of quadruplex formation. In electrophoresis experiments, the quadruplex structure migrates as a single species and more rapidly than the unstructured single strand. NMR spectra in the presence of KCl or NaCl indicate that the structure formed is asymmetric. Equilibrium ultracentrifugation studies confirm that these quadruplexes are composed of two strands of d(G3T4G3). Possible models for this structure are discussed.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

The structures of d(GCAATTGC){sub 2} and its complex with berenil in solution were analyzed by two-dimensional {sup 1}H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10{degree}C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC){sub 2} system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC){sub 2}. The aromatic drug protons are placed within 5 {angstrom} of the H2 proton of both adenines, the H1{prime}, H5{prime}, and H5{double prime} of both thymidines, and the H4{prime}, H5{prime}, and H5{double prime} of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18{degree}C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45{degree}C. Two molecular models are proposed for the berenil-(GCAATTGC){sub 2} complex; onemore » has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.« less

The interaction of chromomycin A3 with the oligodeoxyribonucleotides 1, d(ATGCAT), 2, d(ATCGAT), 3, d(TATGCATA), and 4, d(ATAGCTAT), has been investigated by 1H and 31P NMR. In the presence of Mg2+, chromomycin binds strongly to the three GC-containing oligomers 1, 3, and 4 but not to the CG-containing oligomer 2. The proton chemical shift changes for 1 and 3 are similar, and these DNA duplexes appear to bind with a stoichiometry of 2 drugs:1 Mg2+:1 duplex. The same stoichiometry of 2 drugs:1 duplex is confirmed with 4; however, proton chemical shift changes differ. An overall C2 symmetry is exhibited by the drug complex with 1, 3, and 4. At a molar ratio of 2.0 (drugs:duplex), no free DNA proton NMR signals remain. Two-dimensional nuclear Overhauser exchange spectroscopy (NOESY) of the saturated chromomycin complex with 1 and 3 positions both chromomycinone hydroxyls and the E carbohydrates in the minor groove and provides evidence suggesting that the B carbohydrates lie on the major-groove side. This is supported by several dipolar coupling cross-peaks between the drug and the DNA duplex. Drug-induced conformational changes in duplex 1 are evaluated over a range of NOESY mixing times and found to possess some characteristics of both B-DNA and A-DNA, where the minor groove is wider and shallower. A widening of the minor groove is essential for the DNA duplex to accommodate two drug molecules. This current minor-groove model is a substantial revision of our earlier major-groove model [Keniry, M.A., Brown, S.C., Berman, E., & Shafer, R.H. (1987) Biochemistry 26, 1058-1067] and is in agreement with the model recently proposed by Gao and Patel [Gao, X., & Patel, D. J. (1989a) Biochemistry 28, 751-762].