Highly-compliant, microcable neuroelectrodes fabricated from thin-film gold and PDMS.

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.

An in vivo murine model of low-magnitude oscillatory wall shear stress to address the molecular mechanisms of mechanotransduction--brief report.

OBJECTIVE: Current understanding of shear-sensitive signaling pathways has primarily been studied in vitro largely because of a lack of adequate in vivo models. Our objective was to develop a simple and well-characterized murine aortic coarctation model to acutely alter the hemodynamic environment in vivo and test the hypothesis that endothelial inflammatory protein expression is acutely upregulated in vivo by low-magnitude oscillatory wall shear stress (WSS). METHODS AND RESULTS: Our model uses the shape memory response of nitinol clips to reproducibly induce an aortic coarctation and allow subsequent focal control over WSS in the aorta. We modeled the corresponding hemodynamic environment using computational fluid dynamics and showed that the coarctation produces low-magnitude oscillatory WSS distal to the clip. To assess the biological significance of this model, we correlated WSS to inflammatory protein expression and fatty streak formation. Vascular cell adhesion molecule-1 expression and fatty streak formation were both found to increase significantly in regions corresponding to acutely induced low-magnitude oscillatory WSS. CONCLUSIONS: We have developed a novel aortic coarctation model that will be a useful tool for analyzing the in vivo molecular mechanisms of mechanotransduction in various murine models.

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study.

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(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(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.

Human telomere quadruplex: refolding and selection of individual conformers via RNA/DNA chimeric editing.

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.

Electrostatics dominate quadruplex stability.

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.

Engineering the quadruplex fold: nucleoside conformation determines both folding topology and molecularity in guanine quadruplexes.

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.

Characterization of an unusual folding pattern in a catalytically active guanine quadruplex structure.

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.

Covalent ligation studies on the human telomere quadruplex.

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.

Calculation of ligand-nucleic acid binding free energies with the generalized-born model in DOCK.

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.

Pb EXAFS studies on DNA quadruplexes: identification of metal ion binding site.

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.