Besik Kankia

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements

The interaction of hexamminecobalt(III), Co(NH(3))(6)(3+), with 160 and 3000-8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH(3))(6)(3+) binding up to the molar ratio [Co(NH(3))(6)(3+)]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH(3))(6)(3+)]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH(3))(6)(3+)]/[P] = 0.3. In the case of 3000-8000 bp DNA only two processes were observed: (i) binding up to [Co(NH(3))(6)(3+)]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B-->Psi transition after [Co(NH(3))(6)(3+)]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH(3))(6)(3+) binding on volume and compressibility have been obtained. The binding of Co(NH(3))(6)(3+) to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH(3))(6)(3+): DeltaV = 9 cm(3) mol(-1) and Deltakappa = 33 x 10(-4) cm(3) mol(-1) bar(-1). The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH(3))(6)(3+) and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K+ ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.

Self-dissociative primers for nucleic acid amplification and detection based on DNA quadruplexes with intrinsic fluorescence.

We have developed a new method, quadruplex priming amplification, to greatly simplify nucleic acid amplification and real-time quantification assays. The method relies on specifically designed guanine-rich primers, which after polymerase elongation are capable of spontaneous dissociation from target sites and forming DNA quadruplex. The quadruplex is characterized by significantly more favorable thermodynamics than the corresponding DNA duplexes. As a result, target sequences are accessible for the next round of priming and DNA amplification proceeds under isothermal conditions with improved product yield. In addition, the quadruplex formation is accompanied by an increase in intrinsic fluorescence of the primers, allowing simple and accurate detection of product DNA.

Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3′ polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5′-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NCs observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Interaction of alkaline-earth metal ions with calf thymus DNA. Volume and compressibility effects in diluted aqueous solutions.

The binding of Mg2+, Ca2+, Sr2+ and Ba2+ ions to calf thymus DNA in solutions has been investigated by ultrasonic and densimetric techniques. The obtained parameters, the apparent molar volume, phiV, and the apparent molar adiabatic compressibility, phiK(S), are very sensitive to hydration of investigated molecules. The interaction between the cations and DNA is accompanied by overlapping their hydration shells and consequently releasing the water molecules from hydration shells to bulk state. The change in the hydration is reflected in the measured parameters, phiV and phiK(S). The magnitude of these hydration changes is determined by the position of the cation relative to DNA atomic groups involved in the binding, and thus can characterize the structure of cation-DNA complexes. The values of the dehydration effects of the binding, deltaphiV and deltaphiK(S), correspond to two direct or higher number of indirect contacts between calf thymus DNA and the cations.

Volume and compressibility effects in the formation of metal-EDTA complexes

We used precise measurements of ultrasonic velocity and density to study the complexation of ethylendiaminetetraacetic acid (EDTA) with Mg2+, Ca2+, Sr2+, and Ba2+ at 25‡C and pH 12. From these measurements we obtained the changes in the molar concentration increment of the ultrasonic velocity δA, the apparent molar adiabatic compressibility δKsΦ, and the apparent molar volume δVΦ of complex formation. The hydration contributions δ(AVh) to the volume effect of binding range from 39.6 to 46.6 cm3-mol-1 while the hydration contribution to the adiabatic compressibility change in the binding, δ(δKh), ranges from 103.9X 10-4 to 131.1 X 10-4 cm3-mol-1-bar-1. These data are interpreted in terms of dehydration of interacting molecules,i.e., transfer of water molecules from the hydration shells of cations and EDTA into the bulk water. The ratio δ(δVh)/ δ(δVh) is in the range 0.35 to 0.38 bar, indicating a dominant contribution from the dehydration of charged atomic groups in the volume and the compressibility effects of complex formation.

Volume changes accompanying interaction of ligands with nucleic acids.

Publisher Summary This chapter provides a brief theoretical and experimental description of current techniques for the measurement of volume changes. The chapter illustrates the applicability of this approach for the interaction of ligands with nucleic acids. Specific examples have been provided from data obtained in laboratories; these include ligands with different physical binding modes, the contribution of nucleic acid conformation, bent deoxyoligonucleotides, and the formation of oligomer duplexes with chemically attached ligands. There are several techniques that are currently in use for the measurement of the volume change, ∆V, for an association reaction. Model-dependent techniques are based on the dependence of the equilibrium constant on hydrostatic pressure; model-independent techniques include direct measurements by dilatometry and indirect measurements of the mass and density of reactants and products of a particular reaction. The ∆V value is obtained from the measurements of the mass and the equilibrium density of solutions before and after mixing. The masses are obtained with a microbalance, and the densities are obtained with a magnetic suspension densimeter or with an Anton Paar (Graz Austria) densimeter.

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: Energetics, hydration, sequence, and ionic effects

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross‐links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross‐links. The incorporation of these platinum adducts in a B‐DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature‐dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis‐[Pt(NH3)2{d(GpG}] or cis‐[Pt(NH3)2 {d(ApG}] cross‐links, and their corresponding unmodified duplexes. The platinated duplexes are less stable and unfold with lower TMs (and ΔG°s) in enthalpy‐driven reactions, which indicates a loss of favorable base‐pair stacking interactions. The folding thermodynamics and hydration effects for the first set of decamers containing the d(GpG) cross‐link was investigated by a combination of titration calorimetry, density, and ultrasound techniques. The hydration parameters showed an uptake of structural water by the platinated duplex and a release of electrostricted water by the control duplex. Relative to the unmodified duplex, the folding of the platinated duplex at 20°C yielded a positive ΔΔG° term [and positive ΔΔH‐Δ(TΔS) compensation] and a negative differential volume change. The opposite signs of the ΔΔG° and ΔΔV terms confirmed its uptake of structural water. Further, solvent‐accessible surface areas calculations for a similar pair of dodecamer duplexes indicated that the modified duplex has a 503 œÅ2 higher polar and nonpolar surface area that is exposed to the solvent. Therefore, the incorporation of a platinum adduct in duplex DNA disrupts favorable base‐pair stacking interactions, yielding a greater exposure of aromatic bases to the solvent, which in turn immobilizes structural water. The overall results correlate nicely with the results reported in the available structural data of nuclear magnetic resonance solution studies.

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

Exponential Quadruplex Priming Amplification for DNA-based Isothermal Diagnostics

Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point‐of‐care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self‐dissociate from the primer‐binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 1010‐fold amplification of DNA signal in less than 40 min.