Enzymatic synthesis and 19F NMR studies of 2-fluoroadenine- substituted RNA

Article abstract of DOI:10.1021/ja047556x

The production of isotopically labeled RNA remains critical to current NMR structural studies. One approach to obtain simple NMR spectra is to label with a nucleus that is not naturally occurring in RNA. Fluorine-19 can serve as a sensitive site-specific probe upon incorporation into RNA. Here we report the efficient in vitro enzymatic synthesis of 2-fluoroadenosine-5′-triphosphate and its incorporation into the HIV-2 transactivation region (TAR) of RNA by DNA template-directed transcription using phage T7 RNA polymerase. We provide unequivocal evidence for this ¹⁹F-substituted base analogue capability to selectively interact with uracil, forming 2F-A-U base pairs in RNA. The introduction of a 2-fluoroadenyl substitution is relatively nonperturbing and provides us with uniquely positioned, sensitive NMR reporter groups to monitor structural changes in the local RNA environment. Copyright

Full text of DOI:10.1021/ja047556x

Published on Web 09/02/2004
Enzymatic Synthesis and 19F NMR Studies of 2-Fluoroadenine-Substituted
RNA
Lincoln G. Scott,† Bernhard H. Geierstanger,‡ James R. Williamson,† and Mirko Hennig*,†
Departments of Molecular Biology, Chemistry, and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, MB33, La Jolla, California 92037, and Genomics
Institute of the NoVartis Research Foundation, 10675 John Jay Hopkins DriVe, San Diego, California 92121
Received April 27, 2004; E-mail: mirko@scripps.edu
The production of isotopically labeled nucleotides remains critical
for structural studies of RNA oligonucleotides using nuclear
quently converted to 2F-ATP by sequential action of adenylate
kinase (adk) and creatine phosphokinase (cpk). A key feature of
the synthesis is the use of a dATP as the phosphate donor that was
regenerated using adk, cpk, and an excess of creatine phosphate.
The nucleotide analogue 2F-ATP was easily separated in 90%
isolated yield from the dATP and incorporated into RNA as readily
as ATP by DNA template-directed transcription using phage T7
RNA polymerase.10 A single 40-mL in vitro transcription reaction
yielded 860 nM of 2F-Ade-substituted HIV-2 TAR RNA (see
Supporting Information) that was used for further characterization
by UV and NMR spectroscopy.
To determine the effect of 2F-Ade substitutions on the thermo-
dynamic stability of HIV-2 TAR RNA, optical melting profiles were
performed for 2F-Ade-substituted as well as unmodified RNA
samples (Supporting Information Figure 1). Both RNAs exhibited
essentially identical hyperchromicity in a single transition, with the
Tm for 2F-Ade-substituted RNA being only 2° lower than unmodi-
fied RNA (348.0 ( 0.2 and 350.4 ( 0.7 K, respectively). The
enthalpy values of denaturation for 2F-Ade-substituted and unmodi-
fied RNAs were also quite similar (∆H° ) -92.9 ( 0.7 and
-94.1 ( 1.4 kcal‚mol-1, respectively), clearly demonstrating that
the 19F substitutions do not substantially change overall RNA
stability. Any destabilizing electrostatic interactions in a 2F-A-U
base pair involving positions Ade 19F-2 and Ura O-2 are compen-
sated for via favorable base stacking11 and 19F-N-1 secondary
electrostatic hydrogen-bond donor and acceptor arrangements.12
Apparently, 2F-Ade substitutions are very similar in character to
2,6-diaminopurine substitutions.12,13
The 19F and imino proton resonance assignments for the 2F-
Ade-substituted RNA were straightforward to obtain with homo-
and heteronuclear NOE experiments shown in Figure 1. Two out
of four 19F nuclei, A20 and A27, could be identified on the basis
of intense heteronuclear NOE correlations14,15 to exchangeable
imino protons on the complementary base-paired U42 and U38,
respectively. The exchangeable U42 and U38 imino protons
experience very pronounced upfield chemical shift changes com-
pared to the unmodified RNA (-2.63 and -2.60 ppm, respectively)
because of the altered shielding in the presence of the 19F nuclei
on the complementary base involved in the Watson-Crick base
pair (Supporting Information Figure 2). However, chemical shifts
of guanine imino protons involved in canonical G-C base pairs
are relatively unperturbed (+0.04 ppm on average, Supporting
Figure 2), greatly facilitating the assignments of imino protons in
the 2F-Ade-substituted RNA (Figure 1C). The U40 imino proton
resonance is not detectable at 283 K because of unfavorable
exchange properties with water, and consequently, heteronuclear
NOE correlations to the base-paired 19F nuclei of A22 were not
observed. The 19F resonance of A22 could be unambiguously
assigned using a sequential heteronuclear NOE correlation to the
magnetic resonance (NMR) spectroscopy.1 Through the use of 13
C
and 15N isotopic labeling and multidimensional heteronuclear NMR
experiments, studies of 15-kDa RNAs are commonplace.2,3 How-
ever, as the size of the RNA increases, the spectral crowding and
resonance line widths also increase, making heteronuclear NMR
experiments less sensitive and assignments more difficult. Site-
specific deuteration has been successfully used to simplify spectra
and to improve sensitivity.4 An alternative approach is to label with
a nonnatural nucleus such as fluorine-19 (19F) that affords high
natural abundance and sensitivity. Furthermore, 19F chemical shift
1
dispersion is about 100-fold that of H, making it an ideal site-
specific probe for monitoring functionally important conformational
transitions.5 Here we show that 2-fluoroadenosine-5′-triphosphate
(2F-ATP) can readily be incorporated into the HIV-2 transactivation
response element (TAR) RNA without affecting its structural
integrity and thermodynamic stability. The approach may therefore
be generally applicable to structural studies of RNA molecules.
Previously, we had demonstrated the efficient in vitro enzymatic
synthesis of ribonucleotide-5′-triphosphates (NTPs).4,6,7 The nu-
cleotide analogue 2F-ATP can be produced metabolically in
Escherichia coli supplied with 9-â-D-arabinofuranosyl-2-fluoro-
adenine, which proceeds first by phosphorolysis to 2-fluoroadenine
(2F-Ade), followed by conversion via the purine salvaging pathway
to 2F-ATP.8 We anticipated that 2-fluoroadenine (2F-Ade)-
substituted RNA could be prepared via in vitro enzymatic synthesis
of 2F-ATP,9 followed by in vitro transcription.
The efficient in vitro enzymatic synthesis of 2F-ATP (Scheme
1) began with the C-5-phosphorylation of ribose by ribokinase
Scheme 1
(rbsK) to give ribose-5-phosphate (R5P) that was further phospho-
rylated at the C-1 position by 5-phospho-D-ribosyl-R-1-pyrophos-
phate synthase (prsA), forming 5-phospho-D-ribosyl-R-1-pyrophos-
phate (PRPP). Next, adenine phosphoribosyltransferase (aprT)
coupled 2F-Ade to PRPP, forming 2-fluoroadenosine-5′-mono-
phosphate (2F-AMP). The monophosphate 2F-AMP was subse-
† The Scripps Research Institute.
‡ Genomics Institute of the Novartis Research Foundation.
9
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J. AM. CHEM. SOC. 2004, 126, 11776-11777
10.1021/ja047556x CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
A closer inspection of the 19F spectrum of 2F-Ade-substituted
RNA reveals the presence of minor populations of RNA in addition
to the assigned four major resonances, which can be attributed to
both incomplete 19F labeling and conformational heterogeneity. The
NOESY diagonal peak integrals of the U38 imino proton in the
presence and absence of the 19F nuclei on the complementary base
A27 (Figure 1C) can be attributed to a contamination of 1.3%
unmodified adenine present in the synthesis of 2F-ATP, which was
subsequently incorporated during transcription. Conformational
heterogeneity of fully 2F-Ade-substituted RNA could be due to a
combination of base-pair opening dynamics and equilibria between
interconverting 2F-A-U base-pairing geometries.18
Here we introduced an efficient in vitro enzymatic synthesis of
the fluorinated nucleotide analogue 2F-ATP. We demonstrate the
stable base-pairing interaction of 2F-Ade with uracil in a helical
RNA structure. Both thermal denaturation studies and NMR analysis
suggest that there is only a mild perturbation of the helical RNA
structure by incorporation of multiple 2F-Ade residues. The
introduction of 19F substitution into the adenine bases provides a
uniquely positioned, sensitive NMR reporter to monitor structural
changes in RNA molecules due to conformational changes or ligand
binding.
Acknowledgment. We thank Ms. Edit Sperling for preparing
the enzyme prsA and Dr. Kenneth A. Jacobson of the National
Institutes of Health for insightful discussions. This work was
supported by The Skaggs Institute for Chemical Biology and the
National Institutes of Health (F32 CA80349 to L.G.S.) and (GM-
53757 to J.R.W.).
Figure 1. (A) and (B) Proton-detected 1H, 19F-HOESY (optimized for
detecting exchangeable protons; for details see Supporting Information
Figure 3) showing cross-peaks between 19F nuclei and (A) imino and (B)
anomeric H-1′ protons. The inset in (A) shows a 1-D 19F spectrum with
residue-specific assignments for the four 2-19F adenine resonances. (C) 1H,
1H-NOESY experiment showing imino-imino proton connectivities for the
lower stem (dotted line) and corresponding connectivities for the upper stem
(dash-dotted line). Assignments of individual homonuclear imino-imino
NOESY cross-peaks are given. The diagonal peak marked with an asterisk
represents a minor impurity corresponding to U38 imino proton resonance
frequency in the absence of a 2F-Ade substitution at position A27. (D)
Sequence and secondary structural representation of HIV-2 TAR RNA. The
2F-Ade substitutions are highlighted. Observable 19F-1H-1′ HOESY
correlations (B) are indicated by arrows. 19F spectra (A and B) were recorded
on a three-channel Bruker Avance 400 MHz spectrometer equipped with a
Supporting Information Available: Detailed experimental and
supporting figures for the synthesis, thermal melts, and NMR data. This
material is available free of charge via the Internet at http://pubs.acs.org.
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