Kinetics of DNA refolding from longitudinal exchange NMR spectroscopy

Full text of DOI:10.1002/cbic.201100318

DOI: 10.1002/cbic.201100318
Kinetics of DNA Refolding from Longitudinal Exchange NMR Spectroscopy
Romana Spitzer, Karin Kloiber, Martin Tollinger, and Christoph Kreutz*[a]
Nucleic acids have been attracting a lot of attention due to
the discovery that they can fulfill tasks beyond transforming
the genetic information that is stored in the genome into pro-
teins.[1] Such noncoding ribonucleic acids were discovered a
few years ago, and their function in cellular processes is cur-
rently being addressed by a variety of experimental approach-
es. High-resolution structural studies of riboswitch RNAs, for
example, have provided promising starting points for the de-
velopment of new antibiotics.[2] Notably, the gene regulation
mechanisms of riboswitch RNAs are based on their propensity
to populate alternative folds with (nearly degenerate) free en-
ergies that are modulated in response to external stimuli. Thus,
the ability of a particular RNA sequence to specifically recog-
nize small molecules and to populate multiple folds enables it
to fulfill a gene regulatory function. Notwithstanding, DNA
should be able to accomplish similar tasks as such RNA se-
quences. This hypothesis is supported by the fact that in vitro-
selected DNA enzymes and aptamers with similar or even iden-
tical functionalities as their RNA counterparts are known.[3] Al-
though substantial effort is put into the discovery of naturally
occurring DNA catalysts and aptamers, so far no such species
have been reported. Yet, such DNA-based systems are conceiv-
able and could fulfill a biological function or, on the other
hand, be implemented in a molecular biology toolbox for the
control of gene expression.
groups of thymidines that can be employed to detect and
quantify dynamic properties using relaxation-based NMR tech-
niques for 13CH3-spin systems.[9] Our strategy involves the syn-
thetic preparation of a 13CH3-modified thymidine phosphorami-
dite building block, which is subsequently inserted into DNA
by using standard solid-phase synthesis. We prepared various
13C-methyl labeled variants of a bistable DNA model system to
investigate the kinetics and thermodynamics of the refolding
transition between the two folding states using 13C-longitudi-
nal exchange (ZZ) NMR spectroscopy at various temperatures.
The results are analyzed and discussed in relation to data from
analogous bistable RNA systems.
Two synthesis routes for the preparation of the 13CH3-thymi-
dine labeled phosphoramidite building block 5 have been de-
scribed.[10] Both routes use a lithation step and subsequent
treatment with 13C-methyl iodide to yield the key intermediate.
We basically employed a slight modification of the more
recent synthesis route of Bergstrom et al.[10a] to give the 13C-
modified thymidine nucleoside 3, which was then converted
into the thymidine phosphoramidite 5 using standard proce-
dures (Scheme 1). The synthesis route was started from com-
mercially available 2’-deoxy uridine by treating the hydroxyl
There are some examples of conformational heterogeneity
of DNA. For example, human telomeric repeats are able to co-
exist in multiple G quadruplex forms,[4] which potentially exert
different functions depending on the conformational state of
the quadruplex. In addition, during homologous recombina-
tion, DNA undergoes a strand exchange process to form a Hol-
liday junction. Again, this four-way junction displays conforma-
tional heterogeneity with potential functional implications for
the processing of the Holliday junction during recombination.[5]
While such conformational heterogeneity and secondary struc-
ture rearrangements have been investigated in detail for
RNA,[6] DNA secondary structural heterogeneity was addressed
only recently by using NMR spectroscopic techniques.[7]
NMR spectroscopy has proven to be well suited for detect-
ing and quantifying distinct folding states of nucleic acids, and
for determining the kinetics and the thermodynamics of the
refolding process.[8] In this work we present a site-specific 13C-
isotope labeling procedure for DNA and use it to characterize
secondary structure heterogeneity of a bistable DNA se-
quence.[7] We introduce isolated 13C-spin labels into the methyl
Scheme 1. Synthesis of the 13CH3-modified thymidine phosphoramidite
building block 5. a) tert-Butyldimethylsilyl chloride (2.5 equiv; TBDMS-Cl),
imidazole (5 equiv) in anhydrous dimethylformamide, 508C, 3 h, 100%;
b) sec-BuLi (4.5 equiv), tetramethylenediamine (2.2 equiv; TMEDA), 13C-
methyl iodide (4.0 equiv) in anhydrous tetrahydrofurane, À788C to room
temperature, 2 h, 45%; c) Dowex-50-8w (H+ form) in methanol, room tem-
perature, 36 h, 100%; d) 4,4’-dimethoxytrityl chloride (1.3 equiv; DMT-Cl) in
anhydrous pyridine, room temperature, 3.5 h, 87%; e) (2-cyanoethyl)-N,N-dii-
sopropyl-chlorophosphoramidite (1.25 equiv), ethyldimethylamine (10 equiv)
in anhydrous methylene chloride, room temperature, 3 h, 78%. TBMDS: tert-
butyldimethylsilyl; DMT: 4,4’-dimethoxytrityl; *: 13C.
[a] R. Spitzer, Dr. K. Kloiber, Dr. M. Tollinger, Dr. C. Kreutz
Institute of Organic Chemistry, University of Innsbruck
Innrain 52a, 6020 Innsbruck (Austria)
E-mail: christoph.kreutz@uibk.ac.at
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100318.
ChemBioChem 2011, 12, 2007 – 2010
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2007

groups with the tert-butyldimethylsilyl (TBDMS) protecting
group to give compound 1. The following key step used a lith-
ation procedure with sec-BuLi and treatment with 13C-methyl
iodide to yield the protected precursor 2. Removal of the
TBDMS protecting group yielded the key intermediate 3,
which was converted into the 5’-(4,4’-dimethoxytrityl)-protect-
ed compound 4 and then phosphitylated to give the desired
phosphoramidite building block 5 in good overall yield. Com-
pound 5 was used in standard DNA solid phase synthesis with
average coupling yields greater than 98%. Details on the
chemical synthesis of the 13C-modified phosphoramidite 5 can
be found in the Supporting Information.
The conformational landscapes of nucleic acids can comprise
several local minima with similar free energy differences sepa-
rated by high activation energies. Thus, a biologically function-
al nucleic acid can get trapped in an inactive conformation
and can hardly resolve this misfolded state because of the
high activation barrier. Such folding traps can be found in
large nucleic acids comprising several hundred nucleotides,
like the group I intron.[11] The minimal sequence requirement
that can exhibit such structural polymorphism was determined
by the pioneering work of Micura and co-workers, who de-
signed bistable RNAs.[6d] These systems are able to coexist in
two distinct secondary structures at the same time. Surprising-
ly, the minimal construct comprises only 20 nucleotides, which
implies that such conformational heterogeneity could play a
role in smaller functional RNAs, like microRNAs of comparable
size. Schwalbe and co-workers applied an experimentally
rather demanding real-time NMR spectroscopy approach to
determine the kinetics and the thermodynamics of the refold-
ing process of various RNAs comprising 20 to 35 nucleotides,
which involved the perturbation of the thermodynamic equili-
brium.[6c,i,j] Only recently, we used a sensitive 2’-O-13C-methyl
label to characterize the refolding kinetics and thermodynam-
ics of such bistable RNA systems in unperturbed equilibrium
by longitudinal exchange NMR spectroscopy.[8]
Figure 1. A) Bistable DNA model system 6. Folds 6A and 6B are populated
with a ratio of 7:3 at 298 K. B) 1H,13C HSQC spectrum of the fully 13CH3-thymi-
dine labeled DNA 6. Two resonance sets for the competing secondary struc-
tures 6A and 6B were found and assigned by using reference sequences
(Figures S1 and S2 in the Supporting Information). C) Representative 13C-lon-
gitudinal exchange NMR spectroscopy experiment of selectively T4/T22 la-
beled construct at 309 K and a mixing time of 300 ms. Conditions: 0.8 mm
DNA, 50 mm sodium arsenate buffer, pH 7.0.
bution, which was found to be 7:3 in favor of fold A. We think
that fold A is favored over the competing hairpin motif 6B at
298 K due to the highly stable d(cGNNAg) loop motif.[12] This is
also supported by UV melting curve analysis of reference
sequences 6’ and 6’’ mimicking fold A and B, respectively (see
the Supporting Information). We found a difference in the indi-
vidual melting transition of about 108C between sequence 6’
(fold A mimic, Tm =83.38C) and 6’’ (fold B mimic, Tm =73.68C);
this points to the extra stabilization by the loop sequence.
We then addressed the kinetics and the thermodynamic
properties of the refolding reaction of the bistable DNA 6. For
this purpose, a selectively 13CH3-T4/T22 labeled construct was
used to determine the refolding kinetics from state A to B and
back by 1H-13C ZZ longitudinal exchange NMR spectroscopy
(Figure 1C). While the 13CH3-T4 label could be used to deter-
mine both the forward (kAB) and the backward (kBA) rate con-
stants as a function of temperature, we were able to extract
only the forward rate constant (kAB) from the longitudinal
exchange data of the 13C-methyl label T22 (Table 1). This is due
to significant line broadening of the methyl group resonance
of residue T22 in fold 6B, which originates from dynamics in
the micro- to millisecond time regime, and which results in sys-
Within this work we compare the existing data of bistable
RNAs with a related, rationally designed DNA sequence 6,
which exists in two different stable hairpin folds with nearly
equal population distribution (Figure 1A). This model system
was introduced recently, and its fold heterogeneity and the re-
spective populations were determined by comparative imino
proton NMR and fluorine NMR spectroscopy.[7] Here we use
this system to benchmark our 13C-modified thymidine label as
a means to detect and quantify DNA fold heterogeneity. First,
all five thymidine residues were substituted by the correspond-
ing 13CH3 analogue. As expected, we found ten resonances in a
1
two-dimensional H,13C HSQC spectrum; this indicates slow ex-
change between conformations 6A and 6B on the chemical
shift timescale (Figure 1B). These resonances could be as-
signed to the alternative folds 6A and 6B by using reference
sequences (see the Supporting Information). With six rather
well-resolved peaks at 11.7 T (residues T4, T22 and T23) the
resolution was quite satisfactory, only residues T1 and T14 ex-
hibited nearly degenerate chemical shifts in both dimensions
in the two folds. Peak integration of the best resolved reso-
nance (residue T4) was used to estimate the population distri-
2008
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ChemBioChem 2011, 12, 2007 – 2010

and vice versa, to access folding state 6A starting from 6B
(50%). These data favor a transition state model with base
pairs of both folds being formed and disrupted at the same
time (“associative mechanism”).[8]
Table 1. Refolding kinetics of 26 nt DNA sequence 6 by using longitudi-
nal exchange NMR spectroscopy.
[d]
T
[K]
kAB
]
kBA
]
R1A
]
R1B
KAB
[a]
[b]
[c]
[c]
[sÀ1
[sÀ1
[sÀ1
[sÀ1
]
Refolding kinetics of various bistable RNA systems compris-
ing 20 to 34 nucleotides have been reported.[6b,c,h–j,8] In Ta-
ble S2 in the Supporting Information we compare data from
these systems to our results of the bistable model DNA se-
quence 6. The data indicate a similar kinetic behavior of DNA
and RNA (e.g., RNA S1) of similar size. This suggests that both
nucleic acid species have the inherent propensity to adopt
folds with similar free energies, and to refold between multiple
conformational states under physiological conditions with ex-
Residue T4
306 0.333Æ0.085 0.379Æ0.068 2.47Æ0.38 2.41Æ0.42 0.90Æ0.27
309 0.848Æ0.074 0.632Æ0.048 2.16Æ0.14 2.21Æ0.15 1.35Æ0.14
312 1.40Æ0.14
315 2.79Æ0.54
Residue T22
306 0.380Æ0.055
309 0.889Æ0.054
312 1.68Æ0.13
315 2.46Æ0.47
1.07Æ0.10
1.71Æ0.40
2.05Æ0.24 2.19Æ0.23 1.31Æ0.14
1.69Æ0.76 2.50Æ0.49 1.68Æ0.33
n.d.
n.d.
n.d.
n.d.
2.30Æ0.24
2.13Æ0.13
1.74Æ0.24
1.66Æ0.47
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
change rates that could be of biological relevance (10À1
10 sÀ1).
–
[a] Forward rate constant kAB from longitudinal exchange experiment.
[b] Backward rate constant kBA from longitudinal exchange experiment.
[c] Longitudinal relaxation rates (fold A and B) from longitudinal ex-
change experiments. [d] Equilibrium constant KAB from forward and back-
ward rate constants; n.d.: not determinable due to strong exchange line
broadening of resonance T22B.
We have introduced a native, site-specific labeling procedure
for DNA based on a chemically synthesized 13CH3-modified thy-
midine phosphoramidite. The 13C label was incorporated into a
bistable model system comprising 26 nucleotides, which popu-
lates two hairpin folds with a distribution of 7:3. Fold hetero-
1
geneity was readily detectable in heteronuclear H,13C correla-
tematic underestimation of the corresponding peak volumes
in the ZZ exchange data (see the Supporting Information). The
dynamic process leading to line broadening of resonance T22B
seems to be restricted to the sequence context in fold 6B, and
probably results from the consecutive arrangement of two
“weak” A–T base pairs. Thus, reliable analysis of the longitudi-
nal exchange data of residue T22 (i.e., exchange and longitudi-
nal relaxation rates) was impaired, particularly at higher tem-
peratures.
tion NMR experiments, and by application of 13C-longitudinal
exchange NMR spectroscopy techniques on a specifically la-
beled 13C-T4/T22 DNA construct forward and backward ex-
change rate constants in the temperature range from 306 to
315 K could be obtained. Arrhenius analysis yielded, for the
first time, activation energies and thermodynamic parameters
of the refolding reaction of a bistable DNA sequence. Our re-
sults suggest that DNA, very much like RNA, is able to modu-
late its function by populating alternative secondary structures
at the same time point.
The forward and backward rate constants obtained from res-
idue T4 and the forward rate constants of residue T22 were
then analyzed assuming an Arrhenius temperature depend-
ence; this yielded activation energies as well as thermodynam-
ic parameters for the refolding reaction (Table 2). Using the
Experimental Section
Chemical synthesis and DNA solid phase synthesis: For a de-
tailed description please refer to the Supporting Information.
Table 2. Arrhenius and thermodynamic analysis of the refolding reaction
of bistable DNA 6.
NMR spectroscopy: The NMR samples were prepared by lyophili-
zation of the DNA sodium salt form. The DNAs were dissolved with
sodium arsenate buffer (50 mm), pH 6.5, in H2O/D2O 9:1. NMR data
were acquired on a Varian Inova instrument operating at 11.7 T at
Residue DHAB
[kcalmolÀ1
DSAB
[calmolÀ1
DGAB
[kcalmolÀ1
EaAB
[kcalmolÀ1
EaBA
[kcalmolÀ1
[a]
]
]
]
]
]
T4
11.8
38.6
0.4
46.5Æ10
33.0Æ6.5
1
the temperatures indicated. H NMR spectra of H2O samples were
[a] Calculated for 298 K.
acquired by using
a double-pulsed field gradient spin-echo
(DPFGSE) pulse sequence.[14] Gradient selected phase sensitive
1H,13C HSQC spectra were recorded by using a standard pulse
sequence. Longitudinal exchange experiments are based on pulse
sequences previously published for 15N, yielding 13C, H 2D correla-
1
program Mfold we estimated the helix stabilities of both fold-
Mfold
6 A
Mfold
6 B
ing states, 6A and 6B (DH
=À55.3 kcalmolÀ1 and DH
=
tion maps with amplitude modulation of correlation- and ex-
change-peaks determined by longitudinal exchange rate constants
and the kinetics of interconversion.[8,15] For a sample (0.8 mm) of
sequence 6, arrays of spectra were recorded at 306, 309, 312, and
315 K with mixing periods of 10, 50, 100, 200, 300, 400, 600, and
800 ms (10, 30, 60, 100, 150, 250, 350, 500, 600 and 800 ms at
315 K). The size of the data matrices for each spectrum was 1024ꢁ
48 complex data points, the number of scans was 128 and the
interscan delay was 1.5 s, yielding a total measuring time of 30 h at
each temperature. For a detailed description of data analysis,
please refer to the Supporting Information.
À66.0 kcalmolÀ1).[13] Comparison of these values with the acti-
vation energies of the forward and backward reaction (EAa B
=
46.5 kcalmolÀ1 and EBaA =33.0 kcalmolÀ1, respectively) gives in-
sights into the refolding mechanism of bistable DNA 6. The ac-
tivation barrier (EaAB) to fold from state A to B amounts approxi-
Mfold
6 A
mately to 80% of the helix stability in fold A (DH
). For the
backward reaction a value of about 50% is obtained (EBaA vs.
Mfold
6 A
DH
). This gives an estimate of how many base pairs (80%)
have to be broken up to refold from conformation 6A to 6B,
ChemBioChem 2011, 12, 2007 – 2010
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www.chembiochem.org
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Acknowledgements
Funding by the TWF (project 95421 to C.K.) and FWF (V173 to
K.K.) is acknowledged. C.K. thanks Ronald Micura (Innsbruck),
Bernhard Krꢀutler (Innsbruck), and Robert Konrat (Vienna) for
continuous support and scientific discussions.
Keywords: bistable DNA
spectroscopy · NMR spectroscopy
· DNA · longitudinal exchange
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Received: May 20, 2011
Please note: Minor changes have been made to Table 1 since the publication
of this manuscript in ChemBioChem EarlyView. The Editor.
Published online on July 7, 2011
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