CHAPTER II
Materials and Methods
2.1 Materials 2.1.1 Dyes
The chemical structures of the dyes employed in present work are reported in Fig.
2.1: (a) is a synthetic alkaloid, (b) is an acridine derivative and (c), (d) and (e) are cyanine dyes.
Fig. 2.1.The dyes used in present work.
(e) 10,6-(benzoylamino)-2-{(1Z,3Z)-3-[6- benzoylamino)-3- ethyl-1,3-benzothiazol-2-(3H)-ylidene]-2-methoxyprop-1-
enyl}-31ethy-l.3-benzothiazol-3-ium iodide (T307)
(d) 1-methyl-4-[(3-methyl-2(3H)-
benzothiazolylidene)methyl]methyl sulphate (TO)
N NH2
NH2
HN
N
NH2
NH2
NH
N
S O
N S
HN O O
I
N S
N
(b) 3,6-diamine-9-[6,6-bis(2-aminoethyl)- 1,6-diaminohexyl]acridine (D)
N O
O
O O
Cl-
(a) 8-methyl-2,3,10,11-
tetramethoxydibenzo[a,g]quiinolizinium chloride (Coralyne)
N S
N
(c) 3-methyl-2-[(1-methyl-4(1H)-
pyridinylidene)methyl] methyl sulphate (BO)
Coralyne chloride was from Sigma. The stock solutions were frequently prepared by dissolving weighted amounts of the solid in doubly distilled water.
The acridine derivative called D was synthesized as hydro-bromide salt (D·4HBr) (Bazzicalupi et al., 2007). Stock solutions were prepared by dissolving weighted amounts of the solid in pure DMSO and kept in the dark at -18°C. Working solutions were obtained by dilution of the stock to such a level that the DMSO content could be neglected and were used shortly after preparation.
For ZnD weighted amounts of zinc chloride (Fluka) were dissolved into water and the stock solutions of the metal were standardised by EDTA titration using Eriochrome Black T (ET) as an indicator (Flaschka, 1959). ZnD complexes were obtained by equimolar addition of a ZnCl2 solution to the D solution at pH = 8.0, where the high binding constant ensures complete complex formation.
BO, TO and T307 were synthesised and purified elsewhere (Yarmoluk et al., 2001) and used without further purification. Stock solutions of the dyes were prepared by dissolving weighted amounts of the solid in DMSO and kept in the dark at 4°C. BO and TO solutions were standardised spectrophotometrically, using ε = 3.56×104 M-1cm-1 at λ = 501 nm, I = 0.10 M (NaCl), pH = 7.0 for TO and ε = 5.10×104 M-1cm-1 at λ = 446 nm, I = 0.10 M (NaCl), pH = 7.0 for BO (Yarmoluk et al., 2001). Working solutions were obtained by dilution with water of the stocks to such a level that the DMSO content could be neglected.
All the dye solutions were kept in the dark at 4°C. Concentrations of all employed dyes are expressed in molarity and will be indicated as CD.
2.1.2 Nucleic Acids
Calf thymus DNA (lyophilised sodium salt, highly polymerised) from Pharmacia Biotech (Uppsala, Sweden) was dissolved into water. Stock solutions were standardised spectrophotometrically, using ε = 13200 M-1cm-1 at 260 nm, I = 0.10 M (NaCl), pH = 7.0 as obtained from the sample certificate. CT-DNA was sonicated in order to reduce its length to such a level that polymer orientation contribution to the kinetics could be avoided. DNA sonication was carried out using a MSE-Sonyprep 150 sonicator, by applying to suitable DNA samples (8 mL
at an amplitude of 98µm. The sonicator tip was introduced directly into the solution, this being kept in an ice bath to minimize thermal effects due to sonication. Agarose gel electrophoresis tests indicated that the polymer length was reduced to ca. 800 base pairs. The concentrations of all double stranded polynucleotides are expressed in molarity of base pairs and indicated as CP.
Poly(dA-dT)⋅poly(dA-dT) and poly(dG-dC)⋅poly(dG-dC) lyophilised sodium salts (also from Pharmacia Biotech, Uppsala, Sweden) were directly dissolved into water. Stock solutions of the polynucleotides were standardised spettrophotomertrically using ε = 13200 M-1cm-1 at λ = 262 nm, I = 0.10 M (NaCl), pH = 7.0 for poly(dA-dT)⋅poly(dA-dT) and ε = 16800 M-1cm-1 at λ = 254 nm, I = 0.10 M (NaCl), pH = 7.0 for poly(dG-dC)⋅poly(dG-dC) (Müller and Crothers, 1968; Wells et al., 1970; Schmechel and Crothers, 1971).
Poly(A)⋅poly(U) and Poly(U) lyophilised sodium salts from Sigma were prepared by dissolving weighted amounts in doubly distilled water. Stock solutions of the polynucleotide were standardised spettrophotomertrically using ε = 14900 M-1cm-1 for poly(A)⋅poly(U) at λ = 260 nm, I = 0.10 M (NaCl), pH = 7.0 as obtained from sample certificate and ε = 8900 M-1cm-1 for Poly(U) (Janik, 1971) at λ = 260 nm, I = 0.10 M (NaCl), pH = 7.0.
The solutions of triple helical RNA, poly(A)⋅2poly(U) were prepared by mixing equimolar amounts of poly(A)⋅poly(U) and poly(U) in the appropriate buffer (NaCl 0.1 M, sodium cacodylate ((CH3)2AsO2Na) 0.01 M, pH = 7.0). The concentration of the resulting triplex is given in molarity of base triplets and indicates as CP.
2.1.3 Reaction media
All chemicals were analytical grade reagents and were used without further purification. Measurements of pH were made by a Metrohm (Herisau, Switzerland) 713 pH-meter equipped with a combined glass electrode.
Measurements on dye interactions with different polymers were performed at pH = 7.0 and different ionic strengths, obtained by addition of suitable NaCl amounts. Usually 0.01M NaCac ((CH3)2AsO2Na = Sodium cacodylate) was used as buffer, with the exception of the low ionic strength titrations where lower NaCac buffer concentrations were employed (2 ÷ 3×10-3 M).
The water used to prepare the solutions and as a reaction medium was purified by a Millipore Milli-Q water purification system.
2.2 Methods
2.2.1 Spectrophotometry and spectrofluorometry
Spectrophotometric measurements were carried out on a Perkin Elmer and Co.
Gmbh (Überlingen, Germany) Lambda 35 spectrophotometer and fluorescence measurements were performed on a Perkin Elmer and Co. Gmbh (Überlingen, Germany) LS55 spectrofluorometer. Both apparatuses are equipped with jacketed cell holders, with temperature control to within ± 0.1°C.
Absorbance and fluorescence titrations on DNA/dye systems were performed by adding increasing micro-amounts of the polynucleotide directly into the cell containing the dye solution by means of a syringe connected to a micrometric screw.
The principal component analysis of the dye spectra was performed using the DATAN program developed by Kubista (Kubista et al., 1993).
2.2.2 Circular dichroism
Circular dichroism spectra were recorded on a MOS-450 BioLogic spectrometer (Claix, France). The measurements were performed in 1.0 cm path length cells in the 200-550 nm range.
CD titrations at 25°C were carried out by adding increasing amounts of the dye to a known volume of the polymer solution and recording the spectra after each addition.
2.2.3 Viscosity
The viscosity (η) readings were measured using a Micro-Ubbelohde Viscometer (SCHOTT-GERÄTE Gmbh) whose temperature was controlled by an external thermostat (± 0.1°C).
2.2.4 Kinetics
Stopped-Flow apparatus. The stopped-flow technique enables to measure the rates of reactions in which small volumes of two reactant solutions are rapidly mixed. Small volumes of solutions are driven from high performance syringes through a suitable mixer. The resulting mixture passes through an observation flow cell and is collected into a stopping syringe. Just prior to stopping, a steady state flow is achieved. The solution entering the observation cell is only milliseconds old. The age of this reaction mixture is also known as the dead time
of the stopped-flow system. As the solution fills the stopping syringe, the plunger hits a block, causing the flow to be stopped instantaneously.
Figs. 2.3 and 2.4 show the stopped-flow apparatus assembled in our laboratory.
This instrument allows measuring absorbance or fluorescence changes with time, after the fast mixing of the reactants, and uses a Hi-Tech SF-61 mixing unit connected to the spectrophotometric line by two optical guides. The radiation produced by a suitable lamp, is passed through a Bausch and Lomb 338875 high intensity monochromator and then split into two beams. The reference beam is directly sent to a 1P28 photomultiplier, whereas the measuring beam is sent to the observation chamber through an optical quartz guide, and then, through a second quartz guide, to a second 1P28 photomultiplier. The outputs of the two photomultipliers are balanced before each shot. The kinetic curves are collected by an Agilent (Santa Clara, CA) 54622A storage oscilloscope, transferred to a personal computer and evaluated with the Table Curve program of the Jandel Scientific package (AISN software, Richmond, CA).
The kinetic experiments concerning the D and ZnD complex formation with DNA were performed on a Biologic SFM 300 stopped-flow mixing unit coupled to a spectrophotometric line as described elsewhere (Garcia et al., 2006) using a 405 nm laser as a light source. The acquisition system keeps a record of a number of data points ranging from 10 to 8000 with a sampling interval in the 50 ns to 10 s time scale.
All measurements on dye/polynucleotide systems were carried out under pseudo first-order conditions (CP ≥ 10CD).
Fig. 2.3. HI-TECH Scientific SF-61 mixing unit (front view and rear view respectively).
Fig. 2.4. Internal scheme of a Stopped-flow apparatus.
T-jump apparatus. The T-jump technique allows the rates of reversible reactions with short half-lives (down to microseconds) to be measured. An equilibrated mixture of the reagents is rapidly heated by a short pulse of high voltage current that induces a temperature rise within a few microseconds. If the overall ∆H of the reacting system is different from zero, the system relaxes towards its new equilibrium position and the reaction proceeds until the concentrations have reached their new equilibrium values.
In case of very fast kinetics, below the millisecond time range, the T-jump technique was employed. The T-jump measurements were performed on a home made instrument (Fig. 2.5) with Joule heating, built on the basis of the Riegler et al. prototype (Riegler et al., 1974). The used apparatus is able to work in the absorbance and/or fluorescence mode and both detection modes were employed in the present studies. A tungsten lamp-monocromator system was used as the light source.
Fig. 2.5. Scheme of the T-Jump apparatus used in present work. (1a) lamp, (1b) monochromator, (2) beam splitter, (3) cell, (4) fluorescence photodiode, (5) absorbance photodiode, (6) reference photodiode, (7) spark gap, (8) capacitor, (9) high voltage power supply, (10) digital oscilloscope, (11) computer, (↑) reference/measure voltage balance.
The absorbance and fluorescence signals are both revealed using suitable silica photodiodes (Hamamatsu, S1336, Japan) in place of photomultipliers. The capacitor (0.05 µF) discharge through the measuring cell occurs in about 5 µs and the cell resistance is about 100 Ω. In this time a raise of temperature of 2.5°C is obtained if the capacitor is loaded with 20kV. The kinetic curves are collected by an Agilent (Santa Clara, CA) 54622A oscilloscope, transferred to a PC and evaluated with non-linear least-square fitting procedures performed by a JANDEL (AISN software) program.
Both stopped-flow and T-jump apparatuses are provided with temperature control within ± 0.1°C. For both methods experiment were repeated at least ten times, and the observed spread of time constants was found to range within 10%. The time constants given in this work are average values.
