2.3.1 Preparation of metal complex stock solutions
Five different solvents were used to prepare solutions containing DNA or metal complexes that were used in DNA-binding experiments. The composition of these solvents and the different types of DNA-binding experiments they were used for are presented in Table 2.2. Stock solutions containing 1 mM metal complex were prepared by first dissolving the required quantity of complex in 200 L MeOH, since they did not readily dissolve in water. 10 L of 100 mM HCl solution was added to ensure that the metal complex was fully dissolved, then 790 L of the same solvent used to dissolve the DNA (for a selected experiment) was added. For complex (79), 100% MeOH was used to prepare the stock solution as this complex had limited solubility in the presence of water.
Table 2.2 Composition of different solvents used to prepare stock DNA and metal complex solutions for DNA-binding experiments.
Solvent Composition DNA Experimental
techniques 1 150 mM NH4OAc in MilliQTM
water, pH 7.4
Parallel structures of Q1, c-kit1, Q4
FID, MS titration, CD titration, CD melting, 2 100 mM NH4OAc in MilliQTM
water, pH 7.4 D2, CT-DNA
FID, MS titration, CD titration, CD melting, UV melting, UV titration, 3
100 mM NaCl in MilliQTM water, 15 mM NaH2PO4, 15 mM
Na2HPO4, pH 7.4
Q1 anti-parallel CD titration, CD melting 4
100 mM KCl in MilliQTM water, 15 mM KH2PO4, 15 mM K2HPO4,
pH 7.4
Q1 hybrid-type 1 CD titration 5 100 mM NaCl and 10 mM
LiCaCoa in MilliQTM water, pH 7.4 F21T FRET
a Lithium cacodylate (LiCaCo) was prepared by mixing lithium hydroxide and cacodylic acid at the same concentrations.
65
2.3.2 Purification of oligonucleotides
The sequences of oligonucleotides used in this project and their calculated molecular masses are listed in Table 2.3. The molecular masses of DNA were obtained using the Oligonucleotide Properties Calculator.[202] PCR grade single stranded (ss) oligonucleotides were purchased from Sigma (New South Wales, Australia) as ‘trityl-off’ derivatives and purified using the method reported in previous studies.[58, 154, 203, 204] Dried samples of oligonucleotides (1 mol) were dissolved in 1 mL of 10 mM NH4OAc solution prior to being purified by high performance liquid chromatography (HPLC) using a Waters 1525 Binary HPLC pump together with a C18 (octadecylsilyl) column (8 × 100 mm Waters Delta Pak Radial Cartridge) and a Rheodyne manual injector. The column was equilibrated with 10 mM NH4OAC solution for 30 min prior to the sample being injected, and a linear gradient of 0 – 60% aqueous acetonitrile in 10 mM NH4OAC was used to elute the ssDNA at a flow rate of 1 mL/min over a period of 35 min. Aliquots containing purified ssDNA were collected and combined, then freeze-dried using a Savant SpeedVac (Selby-Biolab, Australia). The solid ssDNA was then redissolved in MilliQTM water and stored at -20 °C prior to further use.
The concentrations of purified oligonucleotides were obtained using the Beer-Lambert Law and the measured absorbance of aqueous solutions at 260 nm.
The extinction coefficients for each type of DNA were determined by adding the values of the component bases, which are 15200, 8400, 12010 and 7050 M-1 cm-1 for guanine, thymine, guanine and cytosine, respectively.[202, 205] As a guide, 5 crude 1 mol samples of DNA supplied from the manufacturer yielded 1 mL ssDNA solution of approximately 1 mM after purification and combination.
66 Table 2.3 Properties of oligonucleotides used in this project.
Annealed
DNA code Description Base sequence 5´- 3´
Molecular Mass (Da)[202]
Molar extinction coefficient (M-1 cm-1)
[202]
Q1
human telomeric single
stranded qDNA GGG(TTAGGG)3 6653.4 240120
F21T human fluorescent
labeled oligonucleotide FAM-GGG(TTAGGG)3-
TAMRA na na
c-kit1
21mer single stranded qDNA
GGG AGG GCG CTG GGA
GGA GGG 6698.4 248250
Q4 four stranded qDNA
(TTG GGG GT)4 9986.8 85250a D2 16-mer double
stranded DNA
d2A GCT GCC AAA TAC CTC C 4786.2 159370 d2B GGA GGT ATT TGG CAG C 4977.3 177370
a The molar extinction coefficient of Q4 was calculated using one strand of DNA, since it was present as ssDNA at the time this measurement was performed.
2.3.3 Preparation of double stranded DNA and quadruplex DNA
The double stranded DNA (dsDNA) molecule D2 was prepared by mixing appropriate amounts of the complimentary ssDNA sequences d2A and d2B together, and then freeze drying the resulting mixture using a Savant SpeedVac. The required volume of solvent 2 (Table 2.2) was then added to the dried sample to give a 1mM solution that was then annealed by heating in a hot water bath for 15 min at 56 °C.
This temperature was selected because it is 10 °C higher than the calculated melting temperature of D2.[58, 202] The resulting stock solution of dsDNA was then allowed to cool gradually to room temperature overnight and stored at -20 °C.
Three ssDNA capable of forming different G-quadruplex structures (Q4, Q1 and c-kit1) were used in this project. In order to prepare a sample of one of these DNA molecules, the requisite amount of ssDNA was first freeze dried using the Savant SpeedVac. The appropriate solvent (Table 2.2) was then added to the resulting solid sample to afford a 1 mM stock solution. In order to obtain a parallel topology, the
67 tetramolecular G-quadruplex Q4 was heated at 90 °C for 15 min and then allowed to cool to 25 °C overnight.[127]
Three different G-quadruplex topologies, namely parallel, anti-parallel and hybrid type 1, (Figure 1.10) were obtained for Q1 by using different solvent (Table 2.2) and annealing conditions. Solutions containing the ssDNA form of Q1 were first annealed at 95 °C for 15 min, and then slowly cooled to room temperature at a rate of 10 °C/hour in order to obtain the parallel G-quadruplex topology.[58] The anti-parallel and hybrid forms of Q1 were obtained by annealing the DNA at 95 °C for 10 min and then cooling in ice for 30 min.[157] Due to its instability at high temperatures, the parallel topology of c-kit1 was obtained by heating a solution of the single stranded form of this DNA at 50 °C for 5 min, and then allowing the solution to cool to room temperature. All annealed DNA samples were kept in a freezer at -20 °C prior to further use.