Site-specific incorporation of nitroxide spin-labels into internal sites of the TAR RNA; structure-dependent dynamics of RNA by EPR spectroscopy [15]

Full text of DOI:10.1021/ja005649i

J. Am. Chem. Soc. 2001, 123, 1527-1528
Site-Specific Incorporation of Nitroxide Spin-Labels
Scheme 1. Preparation of Spin-Labeled RNA
1527
into Internal Sites of the TAR RNA;
Structure-Dependent Dynamics of RNA by EPR
Spectroscopy
Thomas E. Edwards, Tamara M. Okonogi,
Bruce H. Robinson, and Snorri Th. Sigurdsson*
Department of Chemistry, UniVersity of Washington
Seattle, Washington 98195-1700.
ReceiVed September 27, 2000
ReVised Manuscript ReceiVed December 12, 2000
1
label into specific internal, base-paired sites of RNA and analysis
of the spin-labeled RNA by EPR.
RNA can catalyze chemical reactions and interacts in specific
ways with other macromolecules, such as proteins. The mecha-
nistic understanding of RNA function relies on structural informa-
tion; however, solving crystal structures of complex RNA
molecules remains challenging. Therefore, other biophysical
techniques have been used to determine the position of RNA
secondary structure elements (i.e., helices, stem-loops, etc.) in
Nitroxide spin-labels have been incorporated into DNA, by
11
conjugation to either the nucleoside base or the sugar-phosphate
backbone.12 However, the currently available methods for the
incorporation of spin-labels into RNA are restricted to either an
unpaired uridine13 or the 5′-end. Both of these strategies are
somewhat limited because the spin-label cannot be conjugated
to internal, base-paired nucleotides. A variety of molecules have
been conjugated to the 2′-position of base-paired nucleotides in
RNA.15 Particularly attractive is the use of a 2′-amino-modified
RNA, which can be prepared by automated chemical synthesis
using commercially available phosphoramidites (Scheme 1). The
2
14
3
three-dimensional space. For example, fluorescence resonance
energy transfer (FRET) can be used to measure long-range
distances (35-85 Å). Techniques that allow measurement of
intermediate distances would be valuable for improving the
4
resolution of RNA structural models that are derived from FRET
and other biophysical techniques.
2
′-amino group can be reacted with electrophiles, such as aliphatic
Electron paramagnetic resonance (EPR) spectroscopy has been
used to study protein structure and folding, and an EPR
spectroscopic ruler” has been described for measurement of
distances between two unpaired electrons separated by 8-25 Å.
Moreover, EPR has been used to determine the solvent acces-
sibility of spin-labels, making EPR analysis of RNA containing
a single unpaired electron a viable tool for the study of RNA
tertiary structure. EPR is also a sensitive probe of dynamics over
a wide range of motions (ps-ms) and has, for example, been
used to determine the sequence-dependent motion of DNA
duplexes. EPR may, thus, yield information about the role of
dynamics in RNA function, for which there is limited data
16
5
isocyanates.
An isocyanate derivative of tetramethylpiperidyl-N-oxy (TEMPO)
(1) was prepared in one step from the commercially available
“
6
17
4
-amino-TEMPO (Scheme 1). Subsequent reaction of isocyanate
7
1 with 2′-amino-modified RNA gave spin-labeled RNA in >90%
yield (Supporting Information). Electrospray MS and enzymatic
digestion of the modified oligomer verified incorporation of the
spin-label into RNA. HPLC analysis of the enzymatic digest
revealed the absence of a 2′-amino nucleoside and the presence
of a strongly retained substance that was shown to coelute with
the expected spin-labeled nucleoside, prepared by chemical
synthesis (Supporting Information). A nucleoside containing a
reduced spin-label was shown not to be present in the enzymatic
digests, demonstrating that the nitroxide was not reduced during
preparation of 118 or incorporation into RNA. Finally, EPR
spectroscopy of the spin-labeled RNA revealed the presence of a
free radical, as described below.
8
9
available. Furthermore, EPR active probes of dynamics have been
used to elucidate structural differences in local regions within
macromolecules.10 The application of EPR for the study of RNA
structure and dynamics requires incorporation of a stable free
radical. We describe here the incorporation of a nitroxide spin-
*
(
Corresponding author. E-mail: sigurdsson@chem.washington.edu
To investigate the effect of the spin-label on the stability of
RNA secondary structure, we incorporated the spin-label into the
structurally well-characterized trans-activation responsive region
1) For a recent review, see: The RNA World, 2nd ed.; Gesteland, R. F.,
Cech, T. R., Atkins, J. F., Eds.; Cold Spring Harbor: 1999; Vol. 37. Doherty,
E. A.; Doudna, J. A. Annu. ReV. Biochem. 2000, 69, 597-615.
(
2) For recent reviews, see: Doherty, E. A.; Doudna, J. A. Annu. ReV.
Biochem. 2000, 69, 597-615. Holbrook, S. R.; Kim, S. H. Biopolymers 1997,
(11) Spaltenstein, A.; Robinson, B. H.; Hopkins, P. B. Biochemistry 1989,
28, 9484-9495. Strobel, O. K.; Kryak, D. D.; Bobst, E. V.; Bobst, A. M.
Bioconjugate Chem. 1991, 2, 89-95. Miller, T. R.; Alley, S. C.; Reese, A.
W.; Solomon, M. S.; McCallister, W. V.; Mailer, C.; Robinson, B. H.; Hopkins,
P. B. J. Am. Chem. Soc. 1995, 117, 9377-9378.
4
1
1
4, 3-21.
(
3) For a recent review, see: Kjems, J.; Egebjerg, J. Curr. Opin. Biotechnol.
998, 9, 59-65.
(
4) Tuschl, T.; Gohlke, C.; Jovin, T. M.; Westhof, E.; Eckstein, F. Science
994, 266, 785-788. Walter, N. G.; Hampel, K. J.; Brown, K. M.; Burke, J.
(12) Nagahara, S.; Murakami, A.; Makino, K. Nucleosides Nucleotides
1992, 11, 889-901.
M. EMBO J. 1998, 17, 2378-2391. Walter, F.; Murchie, A. I. H.; Lilley, D.
M. J. Biochemistry 1998, 37, 17629-17636.
(
(13) Ramos, A.; Varani, G. J. Am. Chem. Soc. 1998, 120, 10992-10993.
Hara, H.; Horiuchi, T.; Saneyoshi, M.; Nishimura, S. Biochem. Biophys. Res.
Commun. 1970, 38, 305-311.
5) For a recent review, see: Hubbell, W. L.; Cafiso, D. S.; Altenbach, C.
Nat. Struct. Biol. 2000, 7, 735-739.
6) Rabenstein, M. D.; Shin, Y. K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
239-8243. Hustedt, E. J.; Smirnov, A. I.; Laub, C. F.; Cobb, C. E.; Berth,
A. H. Biophys. J. 1997, 72, 1861-1877.
7) Lin, Y.; Nielsen, R.; Murray, D.; Hubbell, W. L.; Mailer, C.; Robinson,
(
(14) Macosko, J. C.; Pio, M. S.; Tinoco, I. Jr.; Shin, Y. K. RNA 1999, 5,
1158-1166.
8
(15) Manoharan, M. In Antisense Research and Applications; Crooke, S.
T., Lebleu, B., Eds.; CRC Press: Boca Raton, Ann Arbor, London, Tokyo,
1993; pp 303-349. Douglas, M. E.; Beijer, B.; Sproat, B. S. Bioorg. Med.
Chem. Lett. 1994, 4, 995-1000. Sigurdsson, S. T.; Tuschl, T.; Eckstein, F.
RNA 1995, 1, 575-583.
(
B. H.; Gelb, M. H. Science 1998, 279, 1925-1929. Ball, A.; Nielsen, R.;
Gelb, M. H.; Robinson, B. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6637-
6
642. Oh, K. J.; Altenbach, C.; Collier, R. J.; Hubbell, W. L. Methods Mol.
Biol. 2000, 145, 147-169.
8) Okonogi, T. M.; Alley, S. C.; Reese, A. W.; Hopkins, P. B.; Robinson,
B. H. Biophys. J. 2000, 78, 2560-2571.
9) For a recent review see: Hagerman, P. J. Annu. ReV. Biophys. Biomol.
Struct. 1997, 26, 139-156.
10) Spaltenstein, A.; Robinson, B. H.; Hopkins, P. B. J. Am. Chem. Soc.
989, 111, 2303-2305.
(16) Sigurdsson, S. T.; Eckstein, F. Nucleic Acids Res. 1996, 24, 3129-
3133.
(
(17) Sigurdsson, S. T.; Seeger, B.; Kutzke, U.; Eckstein, F. J. Org. Chem.
1996, 61, 3883-3884.
(
(18) Nitroxides have been shown to disproportionate in the presence of
HCl, which is generated during the preparation of 1. See: Rozantsev, E. G.
Free Nitroxyl Radicals; Plenum Press: New York, 1970; pp 102-105.
(
1
1
0.1021/ja005649i CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/26/2001

1528 J. Am. Chem. Soc., Vol. 123, No. 7, 2001
Communications to the Editor
the uniform modes of the TAR RNA (Supporting Information).
These results show that there is limited motion of the spin-label
independent of the nucleic acid, a prerequisite for probes of
dynamics. This was not a foregone conclusion, because there is,
in principle, free rotation possible around two bonds in the tether,
namely, those flanking the urea moiety.
Figure 1. The TAR RNA. Four samples were prepared, each containing
one spin-label at position U23, U25, U38 or U40.
The spectra of U23 and U25 show significantly more mobility
(∼40-fold) than spin-labeled U38 or U40. In fact, the mobility
of U23 or U25 is similar to that of a single strand, exhibiting
motion on the nanosecond time scale (Supporting Information).
The finding that nucleotides in a loop are more dynamic than
those located in a duplex region is not unexpected and has been
previously observed in EPR studies of DNA using both partially
Table 1. Thermodynamic Data for Spin-Labeled TAR RNAsa
T
m
-∆G°37
(kcal/mol)
-∆H°
-∆S°
(°C)
(kcal/mol) (cal/mol‚K)
unmodified 50.7 ( 1.1 2.45 ( 0.17 58.2 ( 5.2
180 ( 16
205 ( 26
227 ( 12
167 ( 17
185 ( 10
U23
U25
U38
U40
51.6 ( 1.2 2.93 ( 0.24 66.4 ( 8.4
50.7 ( 0.8 3.12 ( 0.39 73.7 ( 1.8
47.8 ( 0.8 1.78 ( 0.07 53.5 ( 5.3
49.1 ( 1.2 2.19 ( 0.12 59.6 ( 2.8
10
22
flexible and rigid spin-labels. Relaxation experiments using
13
C NMR have also shown increased motion of the bases in the
a
TAR loop.23 While it is possible that some of the differences in
mobility of U23 (or U25) relative to U38 (or U40) are due to
motion of the spin-label independent of the nucleic acid, the EPR
probe clearly distinguishes between nucleotides constrained in a
duplex region and those found in a bulge loop.
Optical melting experiments were performed in 50 mM NaCl, 0.05
EDTA, and 5 mM sodium phosphate buffer (pH 7.0).
mM Na
2
To our knowledge, this is the first EPR study of nucleic acid
dynamics using spin-labels that are tethered to the sugar-moiety
of nucleotides. In contrast to previous EPR studies of nucleic acid
dynamics, this approach does not require a multistep synthesis
1
1
for the site-specific incorporation of the spin-label. The spin-
labeling method is applicable to RNA prepared by solid-phase
synthesis (ca. 40-50 nt long). However, spin-labeled RNA can
be ligated to a much longer RNA that has, for example, been
prepared by in vitro transcription.24 Although this ligation strategy
is not very efficient, it is feasible because small quantities (nmol)
are required for EPR studies.
In conclusion, we have shown that nitroxide spin-labels can
be site-specifically incorporated into internal sites of RNA
utilizing readily available materials. Incorporation of the spin-
label into the TAR RNA does not appreciably affect the stability
of the duplex, indicating that the spin-labels are structurally
nonperturbing. EPR analyses of the spin-labeled TAR RNAs
revealed dramatic structure-dependent mobility differences among
the spin-labeled sites. The changes in mobility of these sites upon
binding to the Tat protein are currently under investigation and
will be reported in due course.
Figure 2. EPR spectra of spin-labeled TAR RNAs at 0 °C in 50%
sucrose/100 mM NaCl, 0.1 mM Na EDTA, and 10 mM sodium phosphate
buffer (pH 7.0). The arrows identify the high and low field extrema of
the spectra. The decreased motion of U38/U40 relative to U23/U25 results
in a wider spectrum.
2
(
TAR) of HIV RNA (Figure 1),19 whose interaction with the Tat
protein is essential for efficient transcription during replication
of the HIV virus.20 Four TAR RNAs were prepared, each
Acknowledgment. We thank NIH (GM56947 and GM55963) and
NIEHS for financial support. T.E.E. was supported by the NIH National
Research Service Award 5 T32 GM08268. We thank members of the
Sigurdsson group and T. Madden for critical reading of the manuscript.
containing a single spin-label in position U23, U25, U38 or U40.
Optical melting experiments were used to determine the thermo-
dynamic parameters of duplex formation for the unmodified TAR
RNA and the spin-labeled RNAs (Table 1).21 The melting curves
for each sample showed a single cooperative and reversible
melting transition. The modified RNAs had melting temperatures
within 3 °C of the unmodified RNA. The only modification that
destabilized the TAR RNA was that of U38, but only by ∼0.7
kcal/mol. Interestingly, modification of both U23 and U25, located
in the bulge loop, stabilized the TAR RNA by ∼0.5 kcal/mol.
These results indicate that the spin-label does not appreciably
perturb the secondary structure of RNA.
Note Added after ASAP. The version of this article posted
ASAP on 1/26/01 contained incorrect data for Tm and -∆G°37
in Table 1 that was introduced during the production process.
The correct version was posted on 2/14/01.
Supporting Information Available: (1) Synthetic procedures for
compound 1 and spin-labeled nucleoside 2, derived from reaction of 1
1
with 2′-amino-2′-deoxyuridine, and their spectra ( H NMR, MS and IR);
The EPR spectra of the spin-labeled TAR RNAs were recorded
in a viscous medium (Figure 2). Under these conditions, the
uniform modes of motion for the duplexes are slow enough for
the spectra to be sensitive to the internal motion of the spin-
label. The spectra for TAR RNA containing spin-labels at U38
and U40 are similar and show the correlation times expected for
(2) protocols for preparation and purification of spin-labeled RNA; (3)
HPLC analyses of spin-labeling reactions and enzymatic digestions of
spin-labeled RNA; (4) EPR spectra of 2, spin-labeled TAR RNA, single
strand and duplex controls; and (5) EPR simulations. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA005649I
(
19) Ippolito, J. A.; Steitz, T. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
819-9824.
20) For recent reviews, see: Karn, J. J. Mol. Biol. 1999, 293, 235-254.
Rana, T. M.; Jeang, K. T. Arch. Biochem. Biophys. 1999, 365, 175-185.
21) Breslauer, K. J. Methods Mol. Biol. 1994, 26, 347-372.
9
(22) Okonogi, T. M.; Robinson, B. H. Manuscript in preparation.
(23) King, G. C.; Harper, J. W.; Xi, Z. J. Methods Enzymol.; 1995; 261,
436-450.
(
(
(24) Moore, M. J.; Sharp, P. A. Science 1992, 256, 992-997.