Formation of RNA Oligomers
A R T I C L E S
activated monomers are proximate and can readily react to form
phosphodiester bonds. The relative orientation of the activated
monomers with RNAs determines whether 3′, 5′- or 2′,
5′-phosphodiester bonds are formed. The stronger binding of
the activated monomers to the montmorillonite results in its more
favorable orientation of the growing RNA for the formation of
3′, 5′ phosphodiester bonds.
Experimental Section
General. Adenosine 5′-monophosphate (AMP), anhydrous Na-
ClO4, cytidine 5′-monophosphate (CMP), 2, 2′-dithiodipyridine,
NaH2PO4, imidazole, potassium hydrogen phthalate (KHP), per-
chloric acid, Trizma base, uridine 5′-monophosphate (UMP) and
trifluoroacetic acid (TFA), were obtained from Sigma. Acetonitrile
(CH3CN) and hydrochloric acid were obtained from Mallinckrodt.
Spectra/Por membrane MWCO: 1000 was obtained from Spectrum
Laboratories, Inc., CA. Anion exchanger resin (10NACNA-38,
OH--Form, type I, Beads 16-50 Mesh), sodium chloride and
magnesium chloride were purchased from J.T. Baker. Montmoril-
lonite H-23 bentonite (Chambers, AZ), H-24 bentonite (Otay, CA,)
H-27 bentonite (Belle Fourche, SD) and H-28 bentonite (Little
Rock, AR) were obtained from Ward’s Natural Science Establish-
ment, Inc., Rochester, NY, via the Department of Earth and
Atmospheric Sciences at Rensselaer Polytechnic Institute.
HPLC analysis was performed on a Hitachi L-7100 pump system
equipped with a Hitachi L-4200 UV-vis detector operating at 260
nm. The negatively charged products were separated on a Dionex
DNA Pac-100 µm (4 × 250 mm) analytical anion exchange column
from Dionex Corporation, Sunnyvale, California, USA using a
gradient of 0 - 0.4 M NaClO4 with 2 mM Tris (pH 8) at a flow
rate of 1 mL/minute. The analysis of samples was also performed
on a reverse phase Alltima C-18, 5 µ (4.6 mm × 250 mm) column
(Alltech) using 3.6% CH3CN in 0.02 M NaH2PO4 with 0.2% TFA,
pH 2.5 (isocratic) as a mobile phase.
Banin Procedure for the Formation of Catalytic Montmo-
rillonites.9 Montmorillonite samples (12 g) were treated with 0.5
M hydrochloric acid (50 mL) by continuous stirring at 4 °C for 30
min. At the end of each treatment excess acid was removed by
centrifugation at 3500 rpm and decanting the supernatant. Fresh
acid (50 mL) was added to the montmorillonite pellet and the
treatment was repeated twice more. H+-montmorillonite was washed
with 100 mL of distilled water at 4 °C for 30 min with constant
stirring. At the end of the washing, excess water was separated by
centrifugation at 3500 rpm and decanting of supernatant. Washing
with water (100 mL) was repeated three more times. The H+-
montmorillonite slurry was added to water (1000 mL) and to this
was added 45 mL of wet anion exchange resin to remove the
residual hydrochloric acid. The mixture was stirred for 30 min, pH
was measured (3.25 ( 0.05) and the anion exchange resin was
removed by filtration. Fresh anion exchange resin was added to a
slurry of the montmorillonite in a beaker of water and it was titrated
with 1 M sodium chloride to pH 6-7 with stirring. The anion
exchange resin was removed by filtration (125 µm) and the
montmorillonite slurry was removed and freeze-dried.
Figure 6. HPLC traces obtained for three representative types of mont-
morillonite. They are Volclay (an excellent catalyst), Little Rock (a good
catalyst) and Chambers (a poor catalyst).
Conclusions
We succeeded in answering the three questions that were
posed in the Objectives. The first question was why was it
necessary to generate catalytic montmorillonites by the Banin
procedure and not by the saturation procedure. The Banin
procedure involves the conversion of the montmorillonite to its
protonated form and then back-titration to pH 6-7 while the
saturation procedure involves treatment of the montmorillonite
with excess sodium chloride. The saturation procedure results
in the replacement of cations in the montmorillonite interlayer
with sodium ions while the Banin procedure only replaces those
protons in the acidic montmorillonite with sodium ions required
to reach pH 6-7. Elemental analyses (data not shown) confirm
that there are fewer sodium ions in the interlayer of the Volclay
when it is titrated to pH 7 than the Otay and Chambers
montmorillonites when they are titrated to pH 6-7, consistent
with the titration data (Figure 5). Titration to pH 6-7 results
in the substitution of only some of the protons with sodium
ions. If most of the sites in the interlayer are filled with sodium
ions it is impossible for the activated monomers to intercalate
between the montmorillonite platelets that catalyze the formation
of RNA oligomers.
The second question is why do only some of the montmo-
rillonites catalyze the oligomerization reactions even though they
were treated by the Banin procedure? When the protonated
montmorillonites of the Otay and Chambers class were titrated
with sodium hydroxide it was observed that significantly larger
volumes of alkali were required to titrate to the pH 7 equivalence
points than was required for the Volclay montmorillonite. This
established that the Otay and Chambers montmorillonites have
more isomorphous substitution of metal ions in the montmo-
rillonite lattice. Consequently there are more sodium ions in
the interlayer between the platelets of the Otay and Chambers
classes of montmorillonites than is present in the interlayer of
the Volclay montmorillonites. The larger amount of cations also
binds the platelets of the Otay and Chambers montmorillonites
together more strongly than Volclay. This makes it impossible
for the activated monomers to intercalate between the clay
platelets so the Otay and Chambers montmorillonites are unable
to catalyze the formation of RNA oligomers.31
The analytical behavior of the sodium montmorillonites when
prepared by the two methods is not unique. For example, an analysis
of a zinc Polkville montmorillonite revealed that the montmorillonite
prepared by the saturation method had about 25% more Zn2+ than
the montmorillonite prepared by the Banin procedure.34
Titration of H+-montmorillonites. One gram of H+-montmo-
rillonite was suspended in 100 mL of water and was titrated with
0.021 M aqueous sodium hydroxide that had been standardized
using KHP.
The answers to questions 1 and 2 made it possible to
determine the reaction pathway, the third question in the
Objectives. The activated monomers react to form oligomers
when they are intercalated between the platelets of the mont-
morillonite. The interlayer provides an environment where the
Preparation of Activated Nucleotides (ImpN). The phospho-
rimidazolides of the 5′-nucleotides were prepared as described
(34) Ferris, J. P.; Hagan, W. J., Jr. Orig. Life EVol. Biosphere 1986, 17,
69–84.
9
J. AM. CHEM. SOC. VOL. 131, NO. 37, 2009 13373