RNA folding and catalysis mediated by iron (II).
ABSTRACT Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.
- Biochemistry 05/1974; 13(9):1841-52. · 3.38 Impact Factor
- Proceedings of the National Academy of Sciences 05/1966; 55(4):941-8. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The evidence showing that the self-assembly of complex RNAs occurs in discrete transitions, each relating to the folding of sub-systems of increasing size and complexity starting from a state with most of the secondary structure, is reviewed. The reciprocal influence of the concentration of magnesium ions and nucleotide mutations on tertiary structure is analyzed. Several observations demonstrate that detrimental mutations can be rescued by high magnesium concentrations, while stabilizing mutations lead to a lesser dependence on magnesium ion concentration. Recent data point to the central controlling and monitoring roles of RNA-binding proteins that can bind to the different folding stages, either before full establishment of the secondary structure or at the molten globule state before the cooperative transition to the final three-dimensional structure.Annual Review of Biophysics and Biomolecular Structure 02/1997; 26:113-37. · 18.96 Impact Factor
RNA Folding and Catalysis Mediated by Iron (II)
Shreyas S. Athavale1,3, Anton S. Petrov1,3, Chiaolong Hsiao2,3, Derrick Watkins2,3, Caitlin D. Prickett2,3, J.
Jared Gossett1,3, Lively Lie1,3, Jessica C. Bowman2,3, Eric O’Neill2,3, Chad R. Bernier2,3, Nicholas V. Hud2,3,
Roger M. Wartell1,2, Stephen C. Harvey1,2,3, Loren Dean Williams2,3*
1School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 2School of Chemistry and Biochemistry, Georgia Institute of Technology,
Atlanta, Georgia, United States of America, 3NAI Center for Ribosomal Origins and Evolution, Georgia Institute of Technology, Atlanta, Georgia, United States of America
Mg2+shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of
essentially all large RNAs. Here we use theory and experiment to evaluate Fe2+in the absence of free oxygen as a
replacement for Mg2+in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments
that suggest that the roles of Mg2+in RNA folding and function can indeed be served by Fe2+. The results of quantum
mechanical calculations show that the geometry of coordination of Fe2+by RNA phosphates is similar to that of Mg2+.
Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4–P6
domain RNA is conserved between complexes with Fe2+or Mg2+. The catalytic activities of both the L1 ribozyme ligase,
obtained previously by in vitro selection in the presence of Mg2+, and the hammerhead ribozyme are enhanced in the
presence of Fe2+compared to Mg2+. All chemical footprinting and ribozyme assays in the presence of Fe2+were performed
under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions.
Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe2+. The
combined biochemical and paleogeological data are consistent with a role for Fe2+in an RNA World. RNA and Fe2+could, in
principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg2+alone.
Citation: Athavale SS, Petrov AS, Hsiao C, Watkins D, Prickett CD, et al. (2012) RNA Folding and Catalysis Mediated by Iron (II). PLoS ONE 7(5): e38024. doi:10.1371/
Editor: Alfred Lewin, University of Florida, United States of America
Received March 19, 2012; Accepted April 28, 2012; Published May 31, 2012
Copyright: ? 2012 Athavale et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding provided by NASA Astrobiology Institute Grant number: NNA09DA78A. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
When large RNAs fold into compact structures, negatively
charged phosphate groups achieve close proximity. Folded RNAs
are stabilized in part by inorganic cations that accumulate in and
around the RNA envelope. ‘Diffuse’ cations remain hydrated and
make primary contributions to global stability by mitigating
electrostatic repulsion of the negatively charged backbone.
Chelated ions are less frequent, but in some instances are essential
for achieving specific local conformation of the RNA. A special
importance of Mg2+in tRNA folding was seen early on [1–3]. It is
now known that Mg2+plays important roles in folding of
essentially all large RNAs [4–6]. In addition, Mg2+ions assist
directly in stabilizing transition states of some ribozymes [7,8].
Here we use computation and experiment to address the
question of whether Fe2+can substitute for Mg2+in RNA folding
and catalysis. Mg2+possesses important electronic and geometric
properties that are key to its relationships with RNA. It is redox
inactive, and does not cleave RNA via Fenton chemistry. The
ionic radius of Mg2+is small, the charge density is high, the
coordination geometry is strictly octahedral, and the hydration
enthalpy is large and negative [9–11]. In comparison with Group I
cations, calcium, or polyamines, Mg2+has a greater affinity for
phosphate oxygens . We find that Fe2+is an excellent Mg2+
mimic in the absence of O2, readily substituting for Mg2+in RNA
folding and catalysis.
Our primary motivation is to study RNA under plausible early
earth conditions. Understanding the influence of Fe2+on RNA
structure and function could provide important links between the
geological record and the RNA world. It is believed that life
originated with RNA-based genetic and metabolic systems, i.e. the
RNA world , which apparently flourished in an anoxic
environment in which iron was much more soluble and abundant
than in our current oxidative environment. Life evolved for
around a billion years before the rise of photosynthesis and the
Great Oxidation Event [14,15]. Fe2+, either instead of or in
combination with Mg2+, seems to be a possible partner of RNA in
the biology of the pre-photosynthesis anoxic earth.
With the rise in free oxygen, a product of photosynthesis, the
Fe2+of the early earth was oxidized and sequestered. Iron was
deposited in banded iron formations (BIFs) , but BIF iron is
seen by isotopic variations to have been a participant in ancient
biological processes . The transition from soluble to insoluble
iron caused slow but dramatic shifts in biology and geology.
Theory predicts that RNA conformation is conserved if
Fe2+substitutes for site-bound Mg2+
Quantum mechanical (QM) calculations show that RNA
conformation and coordination geometry are conserved when
Mg2+is replaced by Fe2+in first shell RNA-metal complexes. We
PLoS ONE | www.plosone.org1May 2012 | Volume 7 | Issue 5 | e38024
focused here on an RNA-Mg2+clamp , in which two adjacent
RNA phosphates coordinate a common Mg2+(Figure 1A). A
complex with multiple first-shell RNA interactions with Mg2+
should provide a stringent test of the ability of Fe2+to substitute.
RNA-Mg2+clamps are common in large RNAs . One
observes twenty-five RNA-Mg2+clamps in the Haloarcula marisortui
large ribosomal subunit , two in the P4-P6 domain of the
Tetrahymena thermophila Group 1 intron , one in a self-splicing
group II intron from Oceanobacillus iheyensis , and one in the L1
ribozyme ligase . The folding and function of each of these
RNAs is Mg2+dependent.
The conformations of an RNA-Mg2+clamp and an RNA-Fe2+
clamp are nearly identical, as determined by Density Functional
Theory (DFT) . The RNA conformation and metal-oxygen
distances and angles are very similar (Figures 1B and 1C). The
calculations do indicate some subtle differences, however. Calcu-
lated interaction energies (energies of complex formation, Table
S1) favor Fe2+over Mg2+by 1.3 kcal/mol in continuum solvent as
indicated by DFT calculations. Natural Bond Orbital analysis 
in the gas phase (Table S2) suggests that more charge is transferred
from phosphate to Fe2+(0.43 e-) than from phosphate to Mg2+
(0.29 e-). This difference implies that compared to Mg2+, Fe2+
better activates the phosphorous of RNA to nucleophilic attack.
The increase in activation is attributable to the accessibility of the
d-orbitals of Fe2+.
Chemical probing suggests that RNA conformation is
conserved when Fe2+substitutes for Mg2+
Selective 29-hydroxyl acylation analyzed by primer extension
(SHAPE) is a powerful RNA foot-printing technique that provides
structural information at single-nucleotide resolution [26–28].
SHAPE has been used to accurately predict and confirm
secondary structures of RNA ranging in length from tRNA to
the HIV-1 genome [27,29]. The method exploits the reactivity of
the 29-hydroxyl groups of RNA to electrophiles to form 29-O-
ribose adducts. Here we employed the SHAPE reagent benzoyl
cyanide (BzCN). The relative reactivities of ribose 29-hydroxyl
groups to the electrophile are sensitive to local RNA flexibility,
which depends primarily on whether or not a nucleotide is base-
paired. Single-stranded nucleotides react preferentially over
double-stranded nucleotides. Reverse transcription using fluores-
cently labeled primers gives products that are truncated at
locations indicating 29-O-ribose adducts. The fragments are
resolved and visualized using capillary electrophoresis. Capillary
electrophoresis data were processed as described .
The secondary structure of the T. thermophila Group I intron P4–
P6 domain was assayed by SHAPE in the presence of Na+alone,
giving a reaction pattern consistent with the known secondary
structure  (Figure 2A). For example, in the stem-loop formed
by residues 143–160, the double-stranded nucleotides of the stem
are unreactive while the GAAA nucleotides of the loop are
reactive. Some of the most reactive nucleotides of the P4–P6
domain secondary structure are located within the A-rich bulge
We probed the structure of the P4–P6 domain RNA in presence
of Mg2+(Figure 2B). The folding of RNAs from secondary
structure to compact native states, containing long-range tertiary
interactions, is known to be Mg2+-dependent [4–6]. The addition
of 2.5 mM Mg2+to the P4–P6 domain RNA causes pronounced
changes in the SHAPE reactivity. SHAPE reactivity increases at
nucleotides 122, 125, 177–179 and 198–200 (indicated by asterisks
in Figure 2B). The Mg2+-dependence of SHAPE reactivities
reflects (i) specific magnesium binding, (ii) diffuse interactions of
Mg2+, and (iii) RNA-RNA tertiary interactions facilitated by Mg2+,
as previously demonstrated for tRNA , RNase P , and
Domain III of the ribosomal large subunit . The pattern of
SHAPE reactivity for P4–P6 domain RNA in the presence of
Mg2+observed here is nearly identical to that described previously
for the same RNA in the presence of Mg2+by Cech and coworkers
(using N-methylisatoic anhydride instead of BzCN) .
The pattern of SHAPE reactivity for P4–P6 domain RNA is
conserved when Mg2+is replaced by Fe2+under anaerobic
conditions. Figure 2C shows that SHAPE reactivities in presence
of 2.5 mM Fe2+are identical, within the accuracy of the
experiment, to those in presence of 2.5 mM Mg2+. These results
suggest that tertiary interactions and even the ‘ion core’ of the P4–
P6 domain are recapitulated by Fe2+in the absence of oxygen. As
expected, if Fe2+is added to the RNA in the presence of
atmospheric free oxygen, the RNA is quickly degraded (not
Figure 1. Conformations of RNA-Mg2+and RNA-Fe2+clamps are nearly identical. A) RNA-Mg2+clamp from the L1 ribozyme ligase (PDB
2OIU). B) RNA-Mg2+clamp optimized by high level QM calculations. C) An optimized RNA-Fe2+clamp. Each cation (Mg2+or Fe2+) is hexacoordinate.
Mg2+is shown as a yellow sphere and Fe2+is shown as a green sphere. Water molecules are omitted from the images for clarity. Distances are in A˚.
RNA Folding and Catalysis Mediated by Iron (II)
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Activity of two ribozymes is enhanced by Fe2+compared
To investigate RNA function in presence of Fe2+, we tested the
catalytic activity of the L1 ribozyme ligase in the presence of Mg2+
or Fe2+(in the absence of oxygen). This ligase catalyzes formation
of a phosphodiester linkage. The 39-hydroxyl group of an RNA
substrate attacks the a-phosphorus of the ribozyme 59-triphos-
phate . This ribozyme was selected in vitro in the presence of
high [Mg2+] (60 mM) by Robertson and Ellington, and has been
described as Mg2+-dependent . The initial rate of ligation in
100 mM Mg2+is 1.461026min21, while the initial rate of ligation
in 100 mM Fe2+is 3.561025min21, which is 25-fold higher
(Figure 3A). A higher rate for Fe2+over Mg2+holds for essentially
any reasonable equimolar concentration of the two cations.
Achieving an equivalent rate of reaction requires around a 10-
fold greater [Mg2+] than [Fe2+]. As expected, this ligase is inactive
in Na+alone. This control, along with the chemical footprinting of
the P4–P6 domain in Na+alone (Figure 2A), confirms the efficacy
of our divalent cation extraction procedure using divalent cation
The hammerhead ribozyme was also assayed for activity in the
presence of Fe2+. Hammerhead ribozyme sequences are widely
distributed in the tree of life . This ribozyme cleaves the RNA
backbone via nucleophilic attack by a 29-hydroxyl group on the 39-
phosphorous atom . In these reactions the initial rate of
hammerhead cleavage in 25 mM Mg2+is 0.011 min21, while the
initial rate of cleavage in 25 mM Fe2+is 0.035 min21, which is 3-
fold higher (Figure 3B). The maximum fraction of cleaved
substrate was about 3-fold greater in Fe2+versus Mg2+. When
100 mM of these two divalent cations were employed, Fe2+again
showed a higher initial rate of cleavage of ,3.5 fold (data not
The results here support a model of early evolution in which
Fe2+was an important metallo-cofactor for RNA. In this model,
Fe2+was replaced over time by Mg2+, by processes driven at least
in part by photosynthesis and the Great Oxidation Event.
The RNA World – on Steroids
The RNA world is hypothesized to have occurred during the
early Archean eon, prior to the Great Oxidation Event. Fe2+in the
early Archean would have been available, soluble and non-toxic.
Our observations here of Fe2+-mediated RNA folding and
catalysis, in combination with paleogeological information, suggest
that RNA could have originated and evolved in association with
Fe2+. The RNA-Fe2+complexes recently observed in extant
biology  could be molecular fossils from the RNA world, akin
to the ribosome. The injection of Fe2+into RNA World models
opens broad new possibilities for ancient biochemistry. RNA and
Fe2+could, in principle, support an array of RNA structures and
catalytic functions far more diverse than RNA with Mg2+alone.
Complexes of RNA with Fe2+offer the prospect of redox
chemistry and electron transfer reactions for ancient ribozymes.
Replacement of Fe2+by Mg2+in RNA is analogous to
replacement of Fe2+by Mn2+in protein enzymes
The conversion of one metal to another is facile in some protein
enzymes. In just one example, Mn2+and Fe2+are used as cofactors
in a broad class of superoxide dismutases (Fe2+/Mn2+SODs) .
The metal cofactors of these SODs can be interconverted between
Mn2+and Fe2+while the coordination geometry, amino acid
sequence and global fold of the protein are conserved. The
discrimination between Fe2+and Mn2+in Fe2+/Mn2+SODs in vivo
is determined by species, organelle, protein isozyme, protein
expression level and metal bioavailability. Metal substitution
appears to be a useful biological strategy in nutrient-limited
environments [37–39]. Falkowski has proposed that during the
Great Oxidation Event, Mn2+was appropriated into some Fe2+
dependent enzymes . Here we suggest that the same strategy
was employed with RNA, where Fe2+was converted to Mg2+. It
has been suggested, based on sites of Fenton cleavage (using iron/
O2), that Fe2+and Mg2+compete for common sites in RNA [40–
42] in vitro.
Figure 2. Addition of Mg2+or Fe2+causes the same changes in the SHAPE reactivity of the P4–P6 domain of the T. thermophila Group
1 intron. A) Shape profile in presence of 250 mM NaCl and no divalent cations. B) The addition of Mg2+increases the reactivity at the sites indicated
with the asterisks and decreases reactivity at other sites. This reaction contains 2.5 mM Mg2+and 250 mM NaCl. C) The addition of Fe2+causes the
same changes in SHAPE reactivity as Mg2+. This reaction contains 2.5 mM Fe2+and 250 mM NaCl.
RNA Folding and Catalysis Mediated by Iron (II)
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QM calculations suggest that an RNA fragment that
forms multiple first-shell interactions with Mg2+does not
change conformation when Mg2is replaced by Fe2+
The metal-oxygen distances and angles are nearly identical in
the Mg2+and Fe2+complexes (Figures 1B and 1C). The QM
calculations do indicate subtle differences between Mg2+and Fe2+
complexes. It appears that more charge is transferred from
phosphate to Fe2+than from phosphate to Mg2+. This electronic
difference, which activates the phosphorus atom to nucleophilic
attack, is attributable to the accessibility of the d-orbitals of Fe2+.
Chemical probing experiments in solution, using the T.
thermophila Group I intron P4–P6 domain RNA,
demonstrate the ability of Fe2+to substitute for Mg2+
during folding of large RNAs
The P4–P6 domain interacts with Mg2+by a complex blend of
diffuse and chelated modes [21,43,44]. In spite of this complexity,
the changes in SHAPE reactivity of RNA induced by association
with Mg2+or Fe2+in the absence of free oxygen are very similar
(Figures 2B and 2C). SHAPE reports local RNA flexibility, which
depends primarily on whether or not a nucleotide is base-paired in
secondary or tertiary interactions. The results for the P4–P6
domain suggest that in the absence of free oxygen, Fe2+can
replace Mg2+in compacting and folding large RNAs. Thus it
appears that Fe2+and Mg2+are nearly interchangeable in their
interactions with RNA.
Fe2+can substitute for Mg2+to support catalysis by
At equimolar concentrations of Mg2+or Fe2+, the initial rate of
ligation observed for the L1 ribozyme ligase is 25-fold higher with
Fe2+than Mg2+in the absence of free oxygen (Figure 3A).
Similarly, at equimolar concentrations of Mg2+or Fe2+, the initial
rate of RNA cleavage observed for the hammerhead ribozyme is
3-fold higher with Fe2+than Mg2+(Figure 3B). In sum, we have
looked at RNA folding in three independent experimental systems,
by chemical footprinting (P4–P6 domain), and with two ribozyme
assays (L1 ribozyme ligase and the hammerhead ribozyme). In
each system examined, Fe2+substitutes for Mg2+in the absence of
free oxygen. The increased activities of the ribozymes with Fe2+
over Mg2+are consistent with our computational results that
suggest Fe2+is slightly better than Mg2+at activating the
phosphorous of RNA to nucleophilic attack. In the hammerhead,
which is one of the best-characterized ribozymes, it has been
shown that a Mg2+ion interacts directly with the scissile phosphate
before and during catalysis . The results here suggest that Fe2+
is a superior substitute for Mg2+in this catalytic role. A variety of
effects such as differential affinity of Fe2+and Mg2+globally, or for
various sites on RNA, could also contribute to differences in
How much Fe2+was available during the time of the RNA
It seems very likely that the early Archean earth provided a
variety of Fe2+-rich microenvironments. On a global scale, the
[Fe2+]marinein the early Archean is subject to debate, and is largely
circumscribed in current models by PCO2(atmospheric pressure of
CO2) in the atmosphere (Fe2+precipitates as siderite: Fe2+CO32).
Early earth PCO2is inferred using estimates of greenhouse effects,
sun luminosity, earth albedo and temperatures required to
maintain liquid oceans. A variety of recent results challenge high
PCO2models [46,47]. If PCO2was low, [Fe2+]marinecould have
been as high as 100–1000 mM [14,48], compared to 0.3–0.8 nM
in the modern ocean . PHS (atmospheric pressure of HS)
would also have been an important influence on [Fe2+]marinedue
to precipitation of FeS .
Figure 3. Ribozyme activity is enhanced by Fe2+compared to
Mg2+. A) L1 ribozyme ligase activity is enhanced in Fe2+compared to
Mg2+. Ligase reactions were performed under anaerobic conditions at
room temperature and 250 mM Na+in 100 mM [Fe2+] or 100 mM [Mg2+].
The reaction components were first annealed in 50 mM HEPES, pH 8.0,
200 mM sodium acetate by incubating at 90uC for 3 min and cooling to
room temperature over 30 min. The ligation reaction was initiated by
adding the appropriate cation salt. The Na+only reaction gave no
product. Reaction progress was monitored by gel electrophoresis. B)
Hammerhead ribozyme activity is enhanced in Fe2+compared to Mg2+.
Hammerhead ribozyme cleavage reactions were performed under
anaerobic conditions at room temperature in 50 mM HEPES, pH 7.5 and
25 mM [Fe2+] or 25 mM [Mg2+]. Substrate and ribozyme RNA strands
were first annealed in 50 mM HEPES buffer by incubating at 90uC for
2 min and cooling to room temperature over 30 min. Cleavage
reactions were initiated by addition of FeCl2 or MgCl2 from stock
solutions. Reactions were monitored by both gel electrophoresis and
capillary electrophoresis, which gave similar results.
RNA Folding and Catalysis Mediated by Iron (II)
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How much Mg2+was available during the RNA World?
The [Mg2+]marine in the early Archean is also uncertain.
Although the models are tentative, it has been suggested that
[Mg2+]marine during this time was attenuated by submarine
hydrothermal systems associated with higher heat flow [51,52],
more vigorous seafloor spreading [53,54], and by reduced Mg2+
delivery to the oceans by smaller continental landmasses or from
the weathering of peridotite in the sea floor . Both of these
phenomena would tend to lower [Mg+2]marineof early oceans in
comparison to today.
Fe2+is the double-edged sword
The early earth’s abundant Fe2+has been oxidized and
sequestered to the extent that current biomass and species
diversity in many ecosystems is limited by Fe2+availability
[56,57]. Iron in the presence of oxygen is rare, toxic, and
biologically expensive to manage [58,59]. Yet living systems are
dependent on and must acquire and utilize iron. The concentra-
tion of iron in cells is on the order of 100 mM, with iron largely
constrained to heme, iron-sulfur clusters, and di-iron or mono-iron
centers, transporters, carriers, exporters, and concentrators such as
ferritins . Because the solubility of ferric iron in water or
plasma is so low (10218M), cells must combat a massive
concentration gradient. The transition from benign and abundant
iron to scarce and toxic iron would have caused a slow but
dramatic shift in biology that required transformations in
biochemical mechanisms and metabolic pathways.
Materials and Methods
The initial atomic coordinates of a Mg2+-RNA clamp were
extracted from the X-ray structure of the H. marismortui large
ribosomal subunit (PDB entry 1JJ2)  as previously described
. The 59 and 39 phosphates were capped with methyl groups in
lieu of the remainder of the RNA polymer and hydrogen atoms
were added, where appropriate. The Fe2+-RNA clamp was
constructed by replacing the magnesium ion with an iron as
The binding of a Mg2+or Fe2+ion to an RNA fragment was
described by the following reactions:
RNA2-+ Me2+(H2O)6 R RNA2--Me2+.N(H2O)4 complex +
where Me2+= (Mg2+, Fe2+)
The reactants and products were fully optimized using density
functional theory (DFT) at the B3LYP level, which combines the
GGA exchange three-parameter hybrid functional developed by
Becke  and the correlation functional of Lee-Yang-Parr 
and the 6–311G(d,p)++basis set and multiplicity=1 as imple-
mented in Gaussian 09 . The Fe2+-RNA clamp and the
Fe2+(H2O)6 were optimized at the unrestricted B3LYP/6–
31G(d,p) level of theory with spin of iron=2 and multiplicity=5.
Single point energies for these complexes were further obtained at
the UB3LYP/6–311++G(d,p) level of theory using SCF options
DIIS, NOVARACC, VTL, MaxCyc=1000.
The interaction energies were calculated in water using the gas
phase optimized geometries within the framework of the Integral
Equation Formalism of Polarized Continuum Model . The
basis set superposition error (BSSE) in the dimer-centered basis set
was obtained by applying the counterpoise procedure of Boys and
Bernardi . The corrected interaction energies were calculated
by taking into account deformational energies of monomers
according to the scheme proposed by van Duijneveldt-van de
Rijdt and van Duijneveldt . The IEFPCM approach was used
to account for the effect of a polar solvent.
Natural Bond Order (NBO)  and Natural Energy Decom-
position Analysis (NEDA) [66,67] calculations were performed on
the optimized complexes at the (U)B3LYP/6–31G(d,p) level of
theory using the GAMESS package .
DNA and RNA synthesis
The genes and RNA transcripts for the L1 ribozyme ligase and
the P4–P6 domain the Tetrahymena thermophila Group 1 intron were
synthesized and purified as described in Text S1. After transcrip-
tion, Mg2+was removed from the RNA by heat annealing in the
presence of divalent cation chelating beads (Hampton Research).
SHAPE reactivity and ribozyme reactions confirm that the
divalent cations are removed by chelating beads.
SHAPE probing of P4–P6 RNA
All manipulations of RNA with Fe2+were conducted in a Coy
chamber with an atmosphere of 85% N2, 10% CO2, 5% H2. P4–
P6 domain RNA (11.25 mg) was lyophilized, transferred to the
anaerobic chamber, left open for several hours, and resuspended
in 240 mL of 50 mM HEPES, pH 8.0, 200 mM sodium acetate
(final concentration) that had been previously deoxygenated by
bubbling with argon for several hours. The RNA was denatured
and renatured using a thermal cycler, by heating to 90uC for
3 min and then quickly cooling to 4uC. Eighty mL of the RNA
solution was aliquoted into three tubes. To the first tube, 10 mL of
25 mM FeCl2 (Avantor Performance Materials) solution was
added. To the second tube, 10 mL of 25 mM MgCl2was added.
To the third tube, 10 mL water was added. The tubes were
incubated at room temperature for 5 minutes. The RNA from
each tube was then divided equally between two additional tubes.
To one tube of each pair, 5 mL of 800 mM benzoyl cyanide
(BzCN) in anhydrous DMSO was added. The other tube of the
pair served as a negative control, to which 5 mL neat DMSO was
added. The benzoyl cyanide reactions are complete in a few
seconds at room temperature . The Fe2+was removed by
treatment with chelating beads. The beads and the associated
divalent cations were removed with a 0.2 micron filtration spin
column. Denaturing SHAPE experiments were performed in
20 mM HEPES pH 8.0 (final concentration) for 4 min at 90uC
using 130 mM N-methylisatoic anhydride (NMIA) in anhydrous
DMSO. Modified RNA was purified using RNeasy Mini Kit
(Qiagen) and re-suspended in 20 mL 16 TE. The recovery after
purification was 65–75%.
A 20-nt long DNA oligomer, 59- GAACTGCATCCATAT-
CAACA -39, that anneals to the 39-end of the P4-P6 domain, was
used to prime reverse transcription. The primer was labeled with
6-FAM at its 59-end (Eurofins MWG Operon). Reverse transcrip-
tion, capillary electrophoresis and data processing were performed
as described .
L1 ribozyme ligation reactions
As noted above, all manipulations in which RNA was in contact
with Fe2+were conducted in a Coy chamber. The substrate RNA
(59-UGCACU-39) labeled with Cy3 at its 59-end and DNA
enhancer (59-GCGACTGGACATCACGAG-39) were purchased
from Eurofins MWG Operon. Aliquoted reaction components (L1
ligase RNA, substrate RNA and DNA enhancer; typical molar
ratio used was 1:0.1:2, respectively) were lyophilized separately,
transferred to the anaerobic chamber, left open for several hours,
and resuspended in 50 mM HEPES, pH 8.0, 200 mM sodium
acetate that had been previously deoxygenated by bubbling with
argon for several hours. The reaction components were annealed
RNA Folding and Catalysis Mediated by Iron (II)
PLoS ONE | www.plosone.org5May 2012 | Volume 7 | Issue 5 | e38024
inside the anaerobic chamber by incubating at 90uC for 3 min and
cooling to room temperature over 30 min. FeCl2was weighed and
dissolved in water inside the anaerobic chamber. The final
volumes of the reaction mixtures were generally 270 mL.
Ligation reactions were initiated by addition of the appropriate
cation salt to the dissolved reaction components. At predetermined
time points, 30 mL aliquots were withdrawn and quenched by
treatment with chelating beads. The beads and divalent cations
were removed with a spin column and the samples were frozen
and stored at 280uC. The L1 ligase RNA was stable for days in
10 mM Fe2+in the anaerobic chamber, but degraded quickly
upon exposure to atmospheric oxygen. After the Fe2+was
removed with chelating beads, the L1 ligase RNA was stable to
exposure to atmospheric oxygen.
For gel analysis, 5 mL of reaction mixture was mixed with 15 mL
loading buffer (8 M urea, 16TTE, 10% glycerol) and denatured
by heating to 90uC for 2 min. The reaction components were then
resolved on 8% denaturing PAGE gels and visualized on a
Typhoon Trio variable mode imager. Some representative gels are
shown in Figure S1. The band intensities were quantified using
Fujifilm MultiGauge 2.0 software.
Hammerhead ribozyme cleavage reactions
As noted above, all manipulations of the hammerhead RNA in
the presence of Fe2+were carried out in a Coy chamber. The
hammerhead ribozyme-substrate was based on the unmodified
HHa1 RNA described by Stage-Zimmermann and Uhlenbeck
.A 31 nucleotidesubstrate
GAAACGCGAAAGCGUCUAGCGGGC-39), labeled at the 39-
end with FAM, and the 21 nucleotide ribozyme strand (59-
CCCGCUACUGAUGAGAUUGCC-39) were purchased from
IDT. Substrate and ribozyme strands (typical molar ratio used was
1:1000) were lyophilized separately, transferred to the anaerobic
chamber, left open for several hours, and resuspended in 50 mM
HEPES, pH 7.5 (pH adjusted with KOH). The buffer had
previously been deoxygenated by bubbling with argon for several
hours. The strands were annealed inside the anaerobic chamber
by incubating at 90uC for 2 min and cooling to room temperature
over 30 min.
Reactions (150 mL final volume) were initiated by addition of
1.5 mL of cation solution (Fe2+or Mg2+). At predetermined time
points, 20 mL aliquots were withdrawn and quenched by
treatment with divalent cation chelating beads. The beads and
the associated divalent cations were removed with a spin column,
and the samples were frozen and stored at 280uC. For gel
analysis, 1 mL of reaction mixture was mixed with 9 mL loading
buffer (8 M urea, 16 TTE, 10% glycerol) and denatured by
heating to 90uC for 2 minutes. The intact 31 nucleotide substrate
and 7 nucleotide product were resolved on 15% denaturing PAGE
gels and visualized on a Typhoon Trio variable mode imager, or
separated by capillary electrophoresis and quantified as described
gels showing L1 Ribozyme Ligase reaction progress.
Only species tagged with 59-Cy3 dye (substrate and product) are
visible. The L1 Ribozyme Ligase is visible when the gel is stained
with cyber gold or ethidium. The reaction rate increases when
[Fe2+] is increased from 100 mM (LH panel, reaction product
observable at 4 hours) to 625 mM (center panel, reaction product
observable at first time point, 30 min). The rate of the reaction in
1 mM Mg2+(RH panel) is roughly equivalent to that in 100 mM
8% polyacryalamide – 8 M urea denaturing
the corresponding counterpoise-corrected interaction
energies calculated at the (U)B3LYP/6–311++
level of theory within the framework of IEFPCM in
Electronic configurations of Mg2+and Fe2+in
the RNA22-Mg2+(H2O)4 and RNA22-Fe2+(H2O)4 com-
plexes as revealed by the NBO at the (U)B3LYP/6–
31G(d,p) level of theory.
Electronic energies, interaction energies and
RNA Synthesis and Purification.
The authors thank Drs. Clark Johnson, Jeffrey Bada, John Peters, Kent
Barefield and Joseph Sadighi for helpful discussions.
Conceived and designed the experiments: SSA ASP CH DW CDP LL
RMW SCH NVH LDW. Performed the experiments: SSA ASP DW CDP
LL. Analyzed the data: SSA ASP DW CDP LL JJG NVH SCH RMW
LDW. Contributed reagents/materials/analysis tools: JCB EO CRB JJG.
Wrote the paper: SSA LDW ASP RMW NVH SCH.
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