19F NMR spectroscopy for the analysis of RNA secondary structure populations

Full text of DOI:10.1021/ja806716s

Published on Web 12/03/2008
19F NMR Spectroscopy for the Analysis of RNA Secondary Structure
Populations
Dagmar Graber, Holger Moroder, and Ronald Micura*
UniVersity of Innsbruck, Institute of Organic Chemistry, Innrain 52a, 6020 Innsbruck, Austria
Received August 27, 2008; E-mail: ronald.micura@uibk.ac.at
RNA offers a functional repertoire that often relies on its intrinsic
conformational flexibility to adopt alternative secondary structure
elements. Several recent examples for RNA dependent regulation
mechanisms can be rationalized by the interplay between the
accessibility of a single-stranded region in one RNA fold, whereas
sequestration in a double helix occurs in the alternative.1,2 In a
molecular perspective, the conformational interplay between single
strand and double helix is reflected by the different conformations
that an oligonucleotide of self-complementary (or partly self-
complementary) sequence can adopt. These conformations are
intermolecular duplexes and intramolecular stem-loop structures
(hairpins) beside unpaired and therefore largely unstructured single
strands.
The original concept was to analyze oligonucleotides of defined
conformational behavior first, on the one hand hairpin systems that
show no competing duplex formation and, on the other hand,
duplexes that are composed of strands whose sequences have no
propensity for hairpin formation. In a second stage, if correlation
of 19F NMR resonances to temperature dependent conformational
changes were successful, oligonucleotides with the inherent pro-
pensity to adopt mixtures of secondary structures would be
analyzed.
An important question of experimental design was the kind of
fluoro labels to be used. Out of a variety of possible sites for NMR
appropriate fluoro labeling of RNA (e.g., ribose 2′-F,5 5-F pyrimi-
dine,6 2-F adenosine,7 5′-CF3 pyrimidine,8a 5′-alkyne-C(CF3)3
pyrimidine8b) we decided in favor of the uracil mimetic 2,4-
difluorotoluene (nucleoside rF).9 The corresponding U to rF
replacements have been recently applied in siRNA approaches and
provided additional motivation to develop a tool with potential
application in the conformational analysis of rF modified siRNAs.10
Figure 1 illustrates the analysis of a short RNA double helix
that is linked by ethylene glycol units on one helix end (Figure
1A). Such RNAs are known for an ideal monomolecular melting
behavior.3e Upon stepwise increase of temperature, the 19F NMR
spectra reflect this behavior by a pronounced shift of the 4-F (Figure
1B,C) and 2-F resonances (Supporting Information) demonstrating
that the melting process occurs in the fast exchange regime on the
NMR time scale. Plotting chemical shift values against temperature
results in a sigmoid curve (Figure 1D). Importantly, the corre-
sponding derivate curve (or alternatively, by applying a sigmoid
fit function) provides the Tm value with good precision for RNA at
concentrations that are generally too high to be accessible for UV
melting analysis. In the present case, the NMR derived Tm value
(low salt concentration) was 2 K lower compared to the UV derived
Tm value (high salt concentration) (Figure 1E). As expected, the
19F NMR-based determination of Tm values is sensitive to varying
salt conditions, and Tm values obtained by the two different methods
under similar salt conditions compare well for the monomolecular,
concentration-independent transition of this RNA hairpin (Sup-
porting Information). The melting behavior of a bimolecular RNA
double helix analyzed by 19F NMR spectroscopy is illustrated in
Figure 2. The sequence of the duplex has been designed for an
ideal two-state transition (Figure 2A). In contrast to the mono-
molecular melting process discussed above, the observed spectra
show that melting of the bimolecular duplex is in the slow exchange
regime on NMR time scale (Figure 2B). Consequently, at temper-
atures close to the melting temperature, two sets of signals are
observed: one for the rF label within the duplex and one for the rF
label within the free single strand. The Tm value can be estimated
by plotting the fraction of single strand against temperature and
then applying a sigmoid curve fit (Figure 2D). The Tm values
obtained by UV melting for this bimolecular melting process
Figure 1. Monomolecular melting transition of a fluorine-labeled RNA:
(A) RNA sequence; (B) 19F NMR spectra at different temperatures; (C)
fluorine-modified nucleobase as uridine replacement; (D) melting profile
derived from chemical shift dependence ∂4F on increasing temperature (cRNA
) 0.3 mM; 25 mM Na2HAsO4, no additional salt, pH 6.5); (E) UV melting
profile (cRNA ) 8 µM; 10 mM Na2HPO4, 150 mM NaCl, pH 7.0).
Even for short oligonucleotides, the experimental verification,
in particular, quantification of the individual conformational popula-
tions can become a complex task.3 Commonly, native gel electro-
phoresis and UV melting profile analysis4 are applied to distinguish
between mono- and bimolecular structures. Here, we demonstrate
that one-dimensional 19F NMR spectroscopy of oligonucleotides
with fluorine labels is a useful and straightforward approach to
obtain direct information on coexisting nucleic acid structures.
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17230 J. AM. CHEM. SOC. 2008, 130, 17230–17231
10.1021/ja806716s CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
compare well when extrapolated to RNA concentrations typical of
NMR studies (Supporting Information).
Having demonstrated the 19F NMR behavior of hairpins and
duplexes, we proceeded with self-complementary sequences that
are prone to exist in a mixture of structures. In the case of 5′-
CGCrFAAUUGGCG we observed a pronounced shift with sigmoid
character for the 2-F 19F resonance indicating hairpin formation
throughout the whole temperature region measured (Figure 3, left).
In the case of 5′-CGCrFAAUUAGCG we observed a distinctly
different signal pattern (Figure 3, right): the constitutional change
of rF:G to rF:A resulted in a second set of 2-F and 4-F resonances
at low temperatures indicating significant duplex over hairpin
competition. The comparison of these two oligonucleotides nicely
exemplifies the convenience of the 19F NMR analysis presented
here. Although basic characterization of such structural equilibria
can be performed by gel shift assays or UV melting profile analysis
the advantage of the 19F NMR approach is direct quantification of
duplex/hairpin populations in a concentration range that is hardly
accessible by the alternative methods.
Figure 3. Structure equilibria of self-complementary RNAs. 19F NMR
spectra at different temperatures; cRNA ) 0.3 mM; 25 mM Na2HAsO4, no
additional salt, pH 6.5; for interpretation see main text.
applications that are incompatible with the decrease in thermody-
namic stability resulting from an A:U to A:rF replacement (Sup-
porting Information).
Acknowledgment. We thank the Austrian Science Fund FWF
for support grant P17864 and Dr. Breuker for advice in error analysis.
Supporting Information Available: Synthesis of rF; analysis of
other rF/dF/5FU RNAs/DNAs; UV melting analysis. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Figure 2. Bimolecular melting transition of a fluorine-labeled RNA: (A)
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19F labeling of RNA with single nonexchangeable fluorine atoms
has become straightforward in recent years and has become an
integrated part of engineered functional RNA with therapeutic
potential, for example, for aptamer, ribozyme, and siRNA technolo-
gies. We have furthermore demonstrated the broad applicability of
the approach for DNA structure equilibria and exemplarily for
another type of fluoro label. The latter is of relevance for specific
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