1.6 Methods for investigating the G-quadruplex DNA binding properties of metal
1.6.1 Electrospray Ionisation Mass Spectrometry (ESI-MS)
Mass spectrometry (MS) is a quantitative technique which can be used to determine the mass of analytes by measuring their mass-to-charge (m/z) ratio.[135]
The fundamental principles of MS were established in late 1890s, after which it became an important method for detecting organic compounds, partially in response to the growing needs of the oil industry in the 1940s.[136] At that time, mass spectrometry was not a widely used method in biochemistry. This was primarily a consequence of the ionisation processes used in mass spectrometers of the day involving high temperatures (200 – 300 °C) and voltages (ca. 500 V). Whilst the volatile mixtures of organic compounds typically analysed by GC-MS methods remained stable under these conditions,[137] biomolecules such as proteins and DNA underwent significant decomposition.[136] It was not until soft ionisation methods such as electrospray ionisation emerged, that it became possible to generate and transfer to the gas phase ions corresponding to biomolecules, and that the potential for analysis of the DNA and proteins by MS methods came to be realised.[138]
Today ESI-MS is a routine method for characterization of proteins and nucleic acids. In addition, it is also suitable for the study of non-covalent binding interactions between these biomolecules and other small molecules, owing to the relatively low temperatures and voltages employed in the ESI process.[139] This has proved very
42 important for enhancing our understanding of biomolecular function, as non-covalent binding interactions with other molecules, including electrostatic, van der Waals, hydrophobic and hydrogen bonding interactions, play important roles in their cellular activities. For example, non-covalent interactions play a part in intermolecular interactions with many binding partners including proteins, lipids, nucleic acids, carbohydrates and small molecules.[138] Importantly it has been shown that non- covalent binding interactions formed between DNA and either metal complexes or small organic molecules remain largely intact during the ESI process. [120, 139, 140]
In addition, ESI-MS also has a number of other favourable attributes for analysis of proteins and nucleic acids, including negligible sample demand and high sensitivity.[141]
ESI mass spectra of metal complexes are typically obtained in positive ion mode owing to the positive charge associated with the metal centres.[142] However, the interactions between metal complexes and DNA are usually investigated in negative ion mode. This is due to the large negative charge on the macromolecule resulting from the many phosphate groups that link the nucleotides which are deprotonated under solution conditions normally used.[143] To obtain mass spectra of solutions containing oligonucleotides it is important to use buffers that do not contain alkali metal cations such as K+ and Na+, as these bind to varying extents to the phosphate groups. This results in noisy ESI mass spectra owing to the method detecting each of the many clusters of ions containing a single oligonucleotide bound to different numbers of cations.[144] In order to avoid this problem, ESI-MS studies involving DNA are usually performed in the presence of volatile salts such as aqueous ammonium acetate.[127, 138, 145] This ensures that the phosphate groups of the oligonucleotides are deprotonated and therefore able to give rise to ions in ESI
43 mass spectra. However, non-covalent adduct formation with the ammonium cation is typically minimal, except when certain G-quadruplex structures are formed. Figure 1.20 shows ESI mass spectra of solutions containing metal complexes and different types of DNA, including two G-quadruplex topologies.
(a)
(b)
(c)
Figure 1.20 Mass spectra of solutions containing different complexes and various DNA molecules obtained on a Q-Tof Ultima ESI-MS instrument: (a) DsDNA and a nickel Schiff base complex;[127] (b) Parallel tetramolecular qDNA and a nickel Schiff base complex;[127]
(c) Parallel unimolecular qDNA and a ruthenium complex.[120] = free DNA; = {DNA + (Ni)}; = {DNA + 2(Ni)}; {DNA + 3(Ni)}; {DNA + 4(Ni)}.
The spectra show two envelopes of ions with different overall charge states.
Within each envelope individual ions are apparent from free DNA and non-covalent adducts containing different numbers of bound metal complex. The spectra show the ability of ESI-MS to provide information about the number, relative amounts and stoichiometry of non-covalent complexes present in these solutions. This can in turn be used to provide information about the relative DNA affinities of a group of related metal complexes or organic compounds towards different DNA molecules. When performing these experiments it must be remembered that the topology of a unimolecular G-quadruplex in a solution containing ammonium acetate may vary
44 from that of the same G-quadruplex in a buffer containing different univalent cations.[145-149] It is therefore essential to also use a technique such as CD spectroscopy, which provides spectra that are highly characteristic of the specific DNA secondary structure present in solution.