Operacionalización de las variables

4.5.1 Mg2+ line broadening and chemical shift analysis of d3'-EBS1* and d3'-

EBS1*·IBS1*

Mg2+ line broadening and chemical shift changes of non-exchangeable protons were monitored by a series of 2D [1H,1H]-NOESY spectra at 298 K with 0.5 mM d3'-EBS1* (100% D2O, I = 10 mM (KCl), 10 µM EDTA, pD = 6.96) and steps of 0, 0.5, 1, 1.5, 2, 2.5, 3,

4, 5, 6, 6.5, and 7 mM MgCl2. The chemical shift map shown in Figure 77 was drawn by

subtraction of the chemical shift of all observed H1', H2, H5, H6 and H8 protons at 0 mM Mg2+ from the corresponding chemical shifts at 2 mM Mg2+. The 2D [1H,1H]-NOESY series for d3'-EBS1*·IBS1* was measured at 298 K with 0.9 mM d3'-EBS1*·IBS1* (100% D2O, I =

110 mM (KCl), 10 µM EDTA, pD = 6.83) and steps at 0, 0.5, 1, 2 3, 4, 5, 6, 7, 8, 10 mM MgCl2. The chemical shift map shown in Figure 77 was drawn by subtraction of the chemical

shift of all observed H1', H2, H5, H6 and H8 protons at 0 mM Mg2+ from the corresponding chemical shifts at 3 mM Mg2+.

Changes in chemical shifts of the imino protons of d3'-EBS1* and d3'-EBS1*·IBS1* were monitored by recording a series of 1D [1H]-NMR experiments, using a spin echo water suppression scheme. In the titration series for d3'-EBS1*, spectra of 0.64 mM RNA (10 mM KCl, 10 µM EDTA, pH = 6.52) in 90% H2O/10% D2O were acquired with 0, 0.5, 1, 2, 3, 4, 5,

6, 7, and 8 mM Mg2+ at 278 K. The chemical shift values at 0 mM Mg2+ were subtracted from the values at 3 mM Mg2+ to yield the chemical shift map of the imino protons shown in Figure 76A. For d3'-EBS1*·IBS1* the titration series was recorded with 0.54 mM RNA (110 mM KCl, 10 µM EDTA, pH = 6.58) and steps at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 25, and 30 mM MgCl2. The chemical shift values at 0 mM Mg2+ were subtracted from the values

at 3 mM Mg2+ to yield the chemical shift map of the imino protons shown in Figure 76B. All spectra were recorded at 700 MHz proton frequency on a Bruker Avance spectrometer equipped with a CP-TXI z-axis pulsed-field gradient cryoprobe.

4.5.2 Mn2+ line broadening studies of d3'-EBS1* and d3'-EBS1*·IBS1*

Mn2+ binding to the non-exchangeable protons of d3'-EBS1* was monitored by titrating MnCl2 in steps of 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 200, 300 µM to a sample of 0.54 mM

d3'-EBS1* (100% D2O, I = 10 mM (KCl), pD = 6.73) and acquisition of 2D [1H,1H]-NOESY

Materials and Methods 171

298 K. In addition a selectively deuterated d3'-EBS1* sample was measured containing 0.6 mM RNA (100% D2O, I = 10 mM (KCl), pD = 6.72) in order to be able to follow also the line

broadening of the signals, which are overlayed by the H5-H6 crosspeaks in non-deuterated samples. The titration steps were 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300 µM Mn2+. Each 2D [1H,1H]-NOESY spectrum was recorded with 48 scans and 2048 experiments in F2 as well as 256 experiments in F1 at 298 K. A 0.6 mM sample of d3'-EBS1*·IBS1* with the d3'-EBS1* hairpin selectively deuterated was used (100% D2O, I = 110 mM (KCl), 20 µM

EDTA, pD = 6.6) for the line broadening experiments with d3'-EBS1*·IBS1*. The 2D [1H,1H]-NOESY series was measured at 298 K with steps of 20, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, and 390 µM MnCl2 (i.e. [Mn2+]tot = 0, 10, 25, 40, 55, 70,

100, 130, 160, 190, 220, 250, 280, 310, 340, and 370 µM due to the binding of Mn2+ to EDTA). Each spectra were recorded with 48 scans and 2048 experiments in F2 as well as 256 experiments in F1.

Mn2+ binding to the imino protons of d3'-EBS1* and d3'-EBS1*·IBS1* was observed by recording 64 scans of a 1D [1H]-NMR series, using a pulse sequence with spin echo water suppression. The sample of d3'-EBS1* contained 0.52 mM RNA (I = 10 mM (KCl), pH = 6.79) and d3'-EBS1*·IBS1* 0.54 mM RNA (I = 110 mM (KCl), pH = 6.89). 1D [1H]-NMR experiments were measured in 90% H2O/10% D2O at 278 K for both samples in steps of 0,

10, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, and 450 µM Mn2+. All spectra were recorded at 700 MHz proton frequency on a Bruker Avance spectrometer equipped with a CP-TXI z-axis pulsed-field gradient cryoprobe. The line broadening effect of Mn2+ coordination to the non-exchangeable as well as to the exchangeable protons was evaluated qualitatively.

4.5.3 Cd2+ titrations - chemical shift analysis of d3'-EBS1* and d3'-

EBS1*·IBS1*

Changes in chemical shifts of the non-exchangeable protons (H1', H2, H6 and H8) of d3'- EBS1* were monitored by 2D [1H,1H]-NOESY spectra upon addition of 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, and 8 mM CdCl2. The d3'-EBS1* sample was selectively deuterated with a

concentration of 0.6 mM (I = 10 mM (KCl), 10 µM EDTA, pD = 6.66). The chemical shift values at 3 mM Cd2+ were subtracted from the values at 0 mM Cd2+ to yield the chemical shift map of the imino protons shown in Figure 101. 2D [1H,1H]-NOESY spectra of d3'- EBS1*·IBS1* were recorded at steps of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, and 8 mM CdCl2.

The concentration of d3'-EBS1*·IBS1* was 0.61 mM (I = 10 mM (KCl), 10 µM EDTA, pD = 6.89). All spectra were measured at 298 K at 700 MHz proton frequency. The chemical shift values at 0 mM Cd2+ were subtracted from the values at 3 mM Cd2+ to yield the chemical shift map of the imino protons shown in Figure 101.

The change in chemical shifts of the exchangeable protons of d3'-EBS1* and d3'- EBS1*·IBS1* was monitored by the acquisition of a series of 1D [1H]-NMR, using a spin echo water suppression scheme. In the titration series of d3'-EBS1*, spectra of a 0.55 mM RNA sample (I = 10 mM (KCl), 10 µM EDTA, pH = 6.72) in 90% H2O/10% D2O were

acquired with 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 25, and 30 mM Cd2+ at 278 K. The d3'-EBS1*·IBS1* sample contained 0.59 mM RNA (I = 110 mM (KCl), 10 µM EDTA, pH = 6.71). In this experiments 1D [1H]-NMR with the same Cd2+ concentration steps like in d3'- EBS1* at 278 K were acquired. The chemical shift maps of the imino protons shown in Figure 99A and B were obtained by subtracting the chemical shift of a certain proton at 0 mM Cd2+ from the value at 2 mM Cd2+. All spectra were acquired at 700 MHz proton frequency.

4.5.4 [Co(NH3)6]

3+

titrations of d3'-EBS1* and d3'-EBS1*·IBS1*

[Co(NH3)6]3+ was titrated to 0.5 mM d3'-EBS1* (I = 10 mM (KCl), 10 µM EDTA, pH =

6.52 in 90% H2O/10% D2O) in steps of 0, 0.5, 1, 1.5, 2, and 2.5 mM. At each titration step 64

scans of a 1D [1H]-NMR experiment with spin echo water suppression were recorded. All experiments were acquired at 278 K and 700 MHz. The chemical shift changes were evaluated by subtracting the chemical shift at 0 mM [Co(NH3)6]3+ from the one at 2 mM

[Co(NH3)6]3+ for each imino proton. At 2.5 mM [Co(NH3)6]3+ a 2D [1H,1H]-NOESY with

watergate H2O suppression was recorded with 64 scans and 256 experiments to observe NOE

crosspeaks between the ammine protons of [Co(NH3)6]3+ and the imino protons of d3'-EBS1*.

For the titration of 0.54 mM d3'-EBS1*·IBS1* (I = 110 mM (KCl), 10 µM EDTA, pH = 6.61 in 90% H2O/10% D2O) 1D [1H]-NMR experiment with spin echo water suppression

were acquired at titration points of 0, 0.5, 1, 1.5, and 2 mM [Co(NH3)6]3+. The resonance line

at 2 mM [Co(NH3)6]3+ were already broadened, thus no titration step with 2.5 mM

[Co(NH3)6]3+ was performed. At 1.5 and 2 mM [Co(NH3)6]3+ 2D [1H,1H]-NOESY

experiments with watergate H2O suppression were recorded with 140 scans and 320

experiments to observe NOE crosspeaks between the ammine protons of [Co(NH3)6]3+ and the

imino of d3'-EBS1*·IBS1*. Acquisition of the spectra was performed at 278 K and in addition for 2 mM [Co(NH3)6]3+ a second 2D [1H,1H]-NOESY spectrum at 283 K was acquired. The

Materials and Methods 173

chemical shift changes were evaluated by subtracting the chemical shift at 0 mM [Co(NH3)6]3+ from the one at 2 mM [Co(NH3)6]3+ for each imino proton.