MAGNESIUM AND CATALYTIC RNA 289
metal ion binding to both the A 9 /G 10.1 and the cleavage site resulted in an
unlikely high - energy state for the ribozyme – substrate complex.^49
The DeRose group also studied hammerhead RNA constructs using para-
magnetic Mn 2+ ions (high - spin d^5 , S = 5/2) examining these with electron para-
magnetic resonance (EPR), electron nuclear double resonance (ENDOR), and
electron spin - echo envelope modulation (ESEEM).^46 These instrumental tech-
niques are discussed in Section 3.5.3. The manganese ion is similar to Mg 2+ in
ionic radius and hydration enthalpy and yields high ribozyme cleavage rates in
0.1 – 1.0 M NaCl. The S = 5/2 Mn 2+ ion has a distinctive six - line EPR pattern that
changes when the ion is bound to RNA, allowing Mn 2+ binding to be quantifi ed.
In addition, the unique signal can be probed for metal coordination details.
One high - affi nity Mn 2+ site was found at 1:1 concentrations of Mn 2+ :RNA
(∼ 100 μ M micromolar) that showed subtle line - shape changes indicating metal
ion – RNA binding. Given this signal the hammerhead metal ion - binding site
could be further examined through hyperfi ne interactions between nuclei of
the RNA ligands and the Mn 2+ electron spin. RNA nuclei with spins that may
be examined in this experiment include the^31 P of RNA phosphodiesters,^1 H
(exchangeable on aqua ligands and non - exchangeable on sugars and bases),
and^14 N on nucleobases. The hyperfi ne interactions can be observed using
ENDOR or ESEEM because both these techniques depend on the infl uence
of nearby nuclear spins on the Mn 2+ EPR signal. Using ENDOR, the research-
ers identifi ed a specifi c high - affi nity, tightly bound Mn 2+ that was coordinated
to a phosphate oxygen (presumably of the A 9 nucleotide) as well as to at least
one aqua ligand. Using ESEEM spectroscopy, the same Mn 2+ was seen to be
coordinated to the N 7 of G 10.1. (See Figure 8 reference 46 .) The DeRose group
also hypothesized that the identifi ed high - affi nity site, populated at micromo-
lar concentrations, must be joined by another weaker binding metal ion site
since at least millimolar concentrations of Mn 2+ in ∼ 1 M NaCl are necessary
to achieve full catalytic activity in the hammerhead. This site is not identifi ed
using EPR studies described in reference 46.
The need for both high - and low - affi nity metal ion - binding sites was echoed
by the hydroxyl radical footprinting experiments carried out by Hampel and
Burke.^56 The footprinting method involves rapid hydroxyl - radical generation
by Fe(II) – EDTA – hydrogen peroxide solutions to study folding kinetics of
RNA complexes. Method details are provided in reference 57 , and a brief
outline is provided here. The solutions containing the RNA constructs to be
studied are incubated with different concentrations of cation, treated with
Fe(II) – EDTA – hydrogen peroxide solutions, and then quenched to stop radical
generation. Hydroxyl radicals are generated according to the following
reaction:
Fe II()−+→−++EDTA H O 22 Fe III( )EDTA iOH OH− (6.3)
Hydroxyl radicals attack the C4 ′ position of the sugar resulting in sugar decom-
position and phosphodiester cleavage. However, if nucleotides are protected