Tercera etapa: Proceso de Diagnóstico en sí y Búsqueda de soluciones

To check the molecular weight of the oligonucleotides, before annealing them to create dsDNA, near native ESI-MS was conducted in negative ion mode, Figure 4.3. The oligonucleotides all produced similar spectra, each displaying a dominant charge series between [M-4H]4- and [M-6H]6-, with little or no fragmentation. In the gas phase, there is the potential for each phosphate group to be deprotonated and to provide the oligonucleotide with a negative charge. The charge states observed, Figure 4.3, were substantially less charged than the oligonucleotide’s theoretical maximum charge. This reduction in charge was proposed to be due to the 10 mM ammonium bicarbonate spraying solution employed. Previous work has indicated that the presence of ammonium bicarbonate/acetate can effect oligonucleotide charge state distributions in a concentration dependent manner (Guo et al. 2005; Touboul and Zenobi 2009). It is thought that ammonium ions, within these buffers, donate protons to the oligonucleotides nitrogenous bases upon entering the gas phase (Guo et al. 2005), therefore reducing the negative charge observed.

166 A B i) i) ii) ii) -4 -4 -5 -5 -6 -6 6726.5 6739.5

After deconvolution onto a true mass scale, both oligonucleotide masses, 6726.5±0.5 and 6739.5±0.5 Da, were in good agreement with their predicted masses of 6726.5 and 6739.5 Da. The smaller peaks present at higher masses in the deconvolution were calculated to be in agreement with the presence of sodium adducts, Figure 4.3.

Figure 4.3: i) Negative ESI mass spectra of A) 5 µM Oligonucleotide one and B) 5µM Oligonucleotide two. ii) shows the deconvolution of the mass spectra.

Once the complementary oligonucleotides had been annealed, to produce dsDNA, containing the S2/S1 SmtB binding site, negative mode ESI-MS experiments were conducted on the oligonucleotide mixture, Figure 4.4. From the mass spectrum it can be seen that there are two different, overlapping, charge state envelopes. One was found to correspond to un-annealed ssDNA, including charge states [M-6H]6- to [M- 3H]3- for both Oligo 1 and Oligo 2. The other charge state envelope corresponded to dsDNA, made up of the charge states [M-8H]8- to [M-6H]6-. The overlap of charge states relating to ssDNA and dsDNA is likely to be more common than the overlap of monomeric and dimeric proteins. As the deprotonation sites within DNA are thought to be the backbone phosphate groups, (Moradian et al. 2002), the formation of a dsDNA helix is unlikely to shield the potential deprotonation sites. In proteins the residues with a potential to become charged are often found to be distributed

167 -7 -3/6 -4/8 -6 -5

evenly across the proteins surface. A percentage of these, on each monomer, will therefore be covered up upon dimerisation.

Figure 4.4: A typical ESI mass spectrum of dsDNA in negative mode, both unbound oligonucleotides and dsDNA with labelled charged states are shown.

The presence of ssDNA oligonucleotides in the annealed sample, Figure 4.4, was unsurprising, since the annealing process between two complementary nucleotides is not 100 % efficient, and no attempt was taken to purify the mixture. The annealing efficiency and stability of dsDNA can be substantially decreased when exposed to low ionic strength. High ionic strengths (100 mM +) were therefore used whenever possible, and diluted prior to MS (Gupta et al. 2001). Here the proportion of C and G bases, within the dsDNA, was found to be low (31 %); which implied a low melting temperature and stability. The breakdown of some dsDNA, into single strands is therefore likely, although attempts were taken to keep this to a minimum.

When compared to the spectra obtained from the oligonucleotide samples, Figure 4.3, a higher proportion of fragmentation in the low m/z range in the dsDNA sample was observed. This is thought to be due to the fragmentation of the ssDNA as the optimum cone voltage for dsDNA was slightly higher than that for ssDNA.

The oligonucleotides used to create the dsDNA had a mass difference of 13 Da and so there is no difficulty assigning m/z peaks for the dsDNA. These range from [M- 6H]6- to [M-8H]8-. If the sequences had been self-complementary, and therefore

168 m/z 1675 1680 1685 1690 1695 % 0 100 1683.8 1680.6 1689.3 m/z 1920 1925 1930 1935 1940 % 0 100 1922.6 1925.7 dsDNA -7 dsDNA -8 Oligo2 -4 Oligo1 -4

possessed the same mass, only odd charge states associated with the duplex could have been confidently assigned.

The mass spectrum of the dsDNA charge state envelope was studied in greater detail to determine if any non-specific association was occurring between the un-annealed ssDNA forming homoduplexes. Low intensity peaks were observed bracketing some of the identified dsDNA peaks, Figure 4.5.

Figure 4.5: Enlarged peaks from the dsDNA charge envelope revealing the presence of Oligo1 and Oligo2, sandwiching the even dsDNA peaks only.

The higher resolution observed on these sandwiching peaks when compared to the known dsDNA peaks, indicated that these peaks were probably due to the presence of lower mass species. This assumption, combined with the observation that the peaks only occurred in conjunction with the [M-6H]6- and [M-8H]8- dsDNA peaks and not the [M-7H]7- dsDNA peak, led to the conclusion that these peaks were probably derived from [M-4H]4- and[M-3H]3- ssDNA oligonucleotides.