Torsion and bending of nucleic acids studied by subnanosecond time-resolved fluorescence depolarization of intercalated dyes

Subnanosecond time‐resolved fluorescence depolarization has been used to monitor the reorientation of ethidium bromide intercalated in native DNA, synthetic polynucleotide complexes, and in supercoiled plasmid DNA. The fluorescence polarization anisotropy was successfully analyzed with an elastic model of DNA dynamics, including both torsion and bending, which yielded an accurate value for the torsional rigidity of the different DNA samples. The dependence of the torsional rigidity on the base sequence, helical structure, and tertiary structure was experimentally observed. The magnitude of the polyelectrolyte contribution to the torsional rigidity of DNA was measured over a wide range of ionic strength, and compared with polyelectrolyte theories for the persistence length. We also observed a rapid initial reorientation of the intercalated ethidium which had a much smaller amplitude in RNA than in DNA

Characterization of ciprofloxacin binding to the linear single- and doublestranded DNA

Abstract The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na + , no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na + , a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g

On the Different Mode of Action of Au(I)/Ag(I)-NHC Bis-Anthracenyl Complexes Towards Selected Target Biomolecules

Gold and silver N-heterocyclic carbenes (NHCs) are emerging for therapeutic applications. Multiple techniques are here used to unveil the mechanistic details of the binding to different biosubstrates of bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride [Ag(EIA)2]Cl and bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) gold chloride [Au(EIA)2]Cl. As the biosubstrates, we tested natural double-stranded DNA, synthetic RNA polynucleotides (single-poly(A), double-poly(A)poly(U) and triple-stranded poly(A)2poly(U)), DNA G-quadruplex structures (G4s), and bovine serum albumin (BSA) protein. Absorbance and fluorescence titrations, mass spectrometry together with melting and viscometry tests show significant differences in the binding features between silver and gold compounds. [Au(EIA)2]Cl covalently binds BSA. It is here evidenced that the selectivity is high: low affinity and external binding for all polynucleotides and G4s are found. Conversely, in the case of [Ag(EIA)2]Cl, the binding to BSA is weak and relies on electrostatic interactions. [Ag(EIA)2]Cl strongly/selectively interacts only with double strands by a mechanism where intercalation plays the major role, but groove binding is also operative. The absence of an interaction with triplexes indicates the major role played by the geometrical constraints to drive the binding mode

The impact of global and local composition on the stability of Triple Helical DNA

It is common practise in antisense technology to view third strand binding to be controlled by the same principles which are found to determine the stability of the double helix. In contrast to this view based on a general consideration of the various forces contributing to the binding energy of the third strand it was proposed that the dominant contributions will originate from electrostatic interactions. These electrostatic contributions can be subdivided into sequence independent repulsive forces between the negatively charged backbones and into sequence dependent attractive forces between the positively charged protonated Hoogsteen cytosines and the backbone phosphates. The observable changes in the stability of triple helices should be a reflection of the number (global composition) and distribution (local composition) of cytosines in the third strand. To this aim two families of 38-mer oligonucleotides were synthesized, which have as a common design feature a linear array of 10 homopurine bases followed by 10 homopyrimidine bases as Watson & Crick complementary strand to the homopurine region and ending in a 10 homopyrimidine residue stretch which binds to the W&C helix via Hoogsteen base-pairing. This arrangement of homopurine and homopyrimidine sections with connecting pyrimidine linkers allows the formation of intramolecular triple helices of predetermined stoichiometry and strand orientation. Physical (UV-spectroscopy, CD-spectroscopy and fluorimetry) and biochemical techniques (P1-nuclease digestion) have been used to show that the oligonucleotides undergo a stepwise folding process from a random coil into a hairpin with 3'dangling tail and then into a intramolecular triple helix. This folding occurs as a function of pH and/or ionic strength. The effect of local and global composition on the stability of the three conformational transitions has been evaluated from a comparison of the melting temperatures and the behavior of the phase boundaries of the different oligonucleotides. As the result of this thesis the following general rules emerge: The stability of the third strand depends on the particular combination of sequence, pH and ionic strength. At physiological conditions (pH 7.1, 150 mM Na⁺) thymines and cytosines contribute equally to the stability (global effect) provided that the cytosines are spaced by more than one thymine. (local effect). Below pH 7.1 (150 mM Na⁺) the stability increases linearly with the number of cytosines and at pH above pH 7.1 ( 150 mM Na⁺) it decreases. At ionic strength below 400 mM Na⁺ (pH 6. 75) the stability increases with the number of cytosine while above 400 mM Na⁺ (pH 6. 75) it decreases. Based on these results a rational approach for the design of oligonucleotide third strands and the choice of appropriate environmental conditions for the formation of a particular triple helix becomes feasible

New aspects of the interaction of the antibiotic coralyne with RNA: coralyne induces triple helix formation in poly(rA)•poly(rU)

The interaction of coralyne with poly(A)•poly(U), poly(A)•2poly(U), poly(A) and poly(A)•poly(A) is analysed using spectrophotometric, spectrofluorometric, circular dichroism (CD), viscometric, stopped-flow and temperature-jump techniques. It is shown for the first time that coralyne induces disproportionation of poly(A)•poly(U) to triplex poly(A)•2poly(U) and single-stranded poly(A) under suitable values of the [dye]/[polymer] ratio (CD/CP). Kinetic, CD and spectrofluorometric experiments reveal that this process requires that coralyne (D) binds to duplex. The resulting complex (AUD) reacts with free duplex giving triplex (UAUD) and free poly(A); moreover, ligand exchange between duplex and triplex occurs. A reaction mechanism is proposed and the reaction parameters are evaluated. For CD/CP> 0.8 poly(A)•poly(U) does not disproportionate at 25°C and dye intercalation into AU to give AUD is the only observed process. Melting experiments as well show that coralyne induces the duplex disproportionation. Effects of temperature, ionic strength and ethanol content are investigated. One concludes that triplex formation requires coralyne be only partially intercalated into AUD. Under suitable concentration conditions, this feature favours the interaction of free AU with AUD to give the AUDAU intermediate which evolves into triplex UAUD and single-stranded poly(A). Duplex poly(A)•poly(A) undergoes aggregation as well, but only at much higher polymer concentrations compared to poly(A)•poly(U)

Localization of metal ions in DNA

M-DNA is a novel complex formed between DNA and transition metal ions under alkaline conditions.  The unique properties of M-DNA were manipulated in order to rationally place metal ions at specific regions within a double-stranded DNA helix.   Investigations using thermal denaturation profiles and the ethidium fluorescence assay illustrate that the pH at which M-DNA formation occurs is influenced heavily by the DNA sequence and base composition.  For instance, DNA with a sequence consisting of poly[d(TG)•d(CA)] is completely converted to M-DNA at pH 7.9 while DNA consisting entirely of poly[d(AT)] remains in the B-DNA conformation until a pH of 8.6 is reached.  The pH at which M-DNA formation occurs is further decreased by the incorporation of 4-thiothymine (s4T).  DNA oligomers with a mixed sequence composed of half d(AT) and the other half d(TG)•d(CA) showed that only 50% of the DNA is able to incorporate Zn2+ ions at pH 7.9.  This suggests that only regions corresponding to the tracts of d(TG)•d(CA) are being transformed.   Duplex DNA monolayers were self-assembled on gold through a Au-S linkage and both B- and M-DNA conformations were studied using X-ray photoelectron spectroscopy (XPS) in order to better elucidate the location of the metal ions.  The film thickness, density, elemental composition and ratios for samples were analyzed and compared.  The DNA surface coverage, calculated from both XPS and electrochemical measurements, was approximately 1.2 x 1013 molecules/cm2 for B-DNA.  All samples showed distinct peaks for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA.  On addition of Zn2+ to form M-DNA the C 1s, P 2p and S 2p showed only small changes while both the N 1s and O 1s spectra changed considerably.  This result is consistent with Zn2+ interacting with oxygen on the phosphate backbone as well as replacing the imino protons of thymine (T) and guanine (G) in M-DNA.   Analysis of the Zn 2p spectra also demonstrated that the concentration of Zn2+ present under M-DNA conditions is consistent with Zn2+ binding to both the phosphate backbone as well as replacing the imino protons of T or G in each base pair.  After the M-DNA monolayer is washed with a buffer containing only Na+ the Zn2+ bound to the phosphate backbone is removed while the Zn2+ bound internally still remains. Variable angle x-ray photoelectron spectroscopy (VAXPS) was also used to examine monolayers consisting of mixed sequence oligomers.  Preliminary results suggest that under M-DNA conditions, the zinc to phosphate ratio changes relative to the position of the d(TG)•d(CA) tract being at the top or bottom of the monolayer.    Electrochemistry was also used to investigate the properties of M-DNA monolayers on gold and examine how the localization of metal ions affects the resistance through the DNA monolayer.  The effectiveness of using the IrCl62-/3- redox couple to investigate DNA monolayers and the potential advantages of this system over the standard Fe(CN)63-/4- redox couple are demonstrated.  B-DNA monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn2+ at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) with IrCl62-/3-.   Compared to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra.  However, only small changes were observed when the monolayers were incubated in Mg2+ at pH 8.6 or in Zn2+ at pH 6.0.  The heterogeneous electron-transfer rate (kET) between the redox probe and the surface of a bare gold electrode was determined to be 5.7 x 10-3 cm/s.  For a B-DNA modified electrode, the kET through the monolayer was too slow to be measured.  However, under M-DNA conditions, a kET of 1.5 x 10-3 cm/s was reached.  As well, the percent change in resistance to charge transfer (RCT), measured by EIS, was used to illustrate the dependence of M-DNA formation on pH.  This result is consistent with Zn2+ ions replacing the imino protons on thymine and guanine residues.  Also, at low pH values, the percent change in RCT seems to be greater for d(TG)15•d(CA)15 compared to oligomers with mixed d(AT) and d(TG)•d(CA) tracts.  The IrCl62-/3- redox couple was also effective in differentiating between single-stranded and double-stranded DNA during dehybridization and rehybridization experiments.

The binding of zinc (II) to a double-stranded oligodeoxyribonucleotide; a voltammetric study.

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62×10-9 to 3.62×10-8 M and 4.06×10-10 to 5.25×10-9 M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation-reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v1/2 (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide

Specific and highly efficient condensation of GC and IC DNA by polyaza pyridinophane derivatives

Two bis-polyaza pyridinophane derivatives and their monomeric reference compounds revealed strong interactions with ds-DNA and RNA. The bis-derivatives show a specific condensation of GC- and IC-DNA, which is almost two orders of magnitude more efficient than the well-known condensation agent spermine. The type of condensed DNA was identified as psi-DNA, characterized by the exceptionally strong CD signals. At variance to the almost silent AT(U) polynucleotides, these strong CD signals allow the determination of GC-condensates at nanomolar nucleobase concentrations. Detailed thermodynamic characterisation by ITC reveals significant differences between the DNA binding of the bis- derivative compounds (enthalpy driven) and that of spermine and of their monomeric counterparts (entropy driven). Atomic force microscopy confirmed GC-DNA compaction by the bis-derivatives and the formation of toroid- and rod-like structures responsible for the psi-type pattern in the CD spectra

Solid-state nanopores: a new platform for DNA biomarker discovery

Solid-state (SS) nanopores emerged as a molecular detection platform in 2001, offering many advantages over their biological counterparts, α-hemolysin nanopores (α-HL). These advantages include better chemical, electrical, mechanical, and thermal stability, as well as size tunability and device integration. In addition, the size of α-HL restricts its application to translocations of single-stranded polynucleotides (ssDNA and ssRNA). This research project focused on developing a SS-nanopore platform for biomarker detection, based on differentiating ssDNA and double-stranded DNA (dsDNA) at the single-molecule scale. Reported dsDNA translocation measurements result in an average residence time of ~ 30 ns/bp, so the temporal resolution required for detection of small DNA duplexes can exceed available bandwidth limitations. To address this issue, several system parameters were explored in order to slow down translocation speed, thereby increasing temporal resolution and signal-to-noise ratio. These parameters included: applied voltage, pH, pore geometry, DNA binding agents, salt composition and concentration, and temperature. Experimental findings showed that SS-nanopores can be precisely fabricated using a controlled helium ion milling technique, acidic conditions cause DNA depurination that results in slower translocation durations, and single-stranded binding proteins (SSBs) bind preferentially to ssDNA, forming complexes with distinct translocation characteristics that permit large (> 7 kb) ds- and ssDNA to be effectively distinguished. Together, these data show that SS-nanopores can serve as a tool to electronically detect the presence and relative concentration of target DNA molecules with ultrahigh sensitivity, thus demonstrating their potential utility as a biomarker discovery platform in both biomedical and environmental applications