Fluorescent ribonucleoside as a FRET Acceptor for tryptophan in native proteins

Article abstract of DOI:10.1021/ja105244t

A new fluorescent ribonucleoside analogue, containing 5-aminoquinazoline-2, 4(1H,3H)-dione, acts as a Foerster resonance energy transfer acceptor for tryptophan (R₀ = 22 A) and displays visible emission (440 nm). As tryptophan is frequently found at or near the recognition domains of RNA binding proteins, this FRET pair facilitates the study of RNA binding to native proteins and peptides, which is demonstrated here for the HIV-1 Rev association with the Rev Response Element (RRE).

Full text of DOI:10.1021/ja105244t

Published on Web 08/09/2010
Fluorescent Ribonucleoside as a FRET Acceptor for Tryptophan in Native
Proteins
Yun Xie, Tucker Maxson, and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093
Received June 15, 2010; E-mail: ytor@ucsd.edu
Scheme 1. Heterocycle 1, Ribonucleoside 2, and the
Corresponding Phosphoramidite 3a
Abstract: A new fluorescent ribonucleoside analogue, containing
5-aminoquinazoline-2,4(1H,3H)-dione, acts as a Fo¨rster reso-
nance energy transfer acceptor for tryptophan (R0 ) 22 Å) and
displays visible emission (440 nm). As tryptophan is frequently
found at or near the recognition domains of RNA binding proteins,
this FRET pair facilitates the study of RNA binding to native
proteins and peptides, which is demonstrated here for the HIV-1
Rev association with the Rev Response Element (RRE).
While tryptophan is one of the most infrequently occurring amino
acids in proteins,1 it is recurrently found at, or near, the recognition
domains of RNA-binding proteins.2,3 As such recognition phenomena
are crucial for transcriptional regulation, mRNA maturation, RNA
interference, translation, and more, the development of biophysical tools
for evaluating these fundamental processes is of key importance.
Although the indole chromophore imparts useful emissive properties,4
tryptophan’s emission is highly sensitive to diverse environmental
perturbations,5 a feature that, while useful, can significantly complicate
the readout and interpretation of binding events. Fo¨rster resonance
energy transfer (FRET) pairs can overcome many of such difficulties,
but the incorporation of large “classical FRET” chromophores into
the binding partners can perturb these delicate recognition events. We
have therefore sought to develop a new isomorphic ribonucleoside
analogue that could seamlessly be incorporated into RNA and serve
as a FRET acceptor to tryptophan residues residing on RNA binding
proteins. Here we report the design, synthesis, photophysical charac-
teristics, and implementation of an emissive uridine mimic that fulfills
such stringent requirements and facilitates the study of RNA-protein
interactions.
a Reagents: (a) (i) N,O-bis(trimethylsilyl)acetamide, CF3SO3Si(CH3)3,
ꢀ-D-ribofuranose 1-acetate 2,3,5-tribenzoate, CH3CN; (ii) conc. NH4OH,
81%. (b) (i) (CH3)3SiCl, phenoxyacetic anhydride, H2O, conc. NH4OH,
pyridine, 75%; (ii) DMTrCl, Et3N, pyridine, 82%; (iii) iPr2NEt, nBu2SnCl2,
iPr3SiOCH2Cl, ClCH2CH2Cl, 30%; (iv) iPr2NEt, (iPr2N)P(Cl)O-
CH2CH2CN, ClCH2CH2Cl, 60%.8
A fundamental challenge in developing minimally perturbing
nucleoside analogues is the tendency of small aromatic chromophores
to display high emission energies, which frequently overlap with the
emission bands of native amino acids.4 A molecular feature that can
help alleviate such constraints and facilitate FRET pairing with Trp is
the implementation of a charge-transfer character upon the chro-
mophore, a mechanism frequently found in naturally occurring visibly
emissive fluorophores (e.g., oxyluciferin and GFP).4 Heterocycle 1
and the corresponding ribonucleoside 2 (Scheme 1), containing an
electron-rich ring fused into the electron-deficient pyrimidine, fulfill
this requirement and display red-shifted absorption and emission bands.
The extinction coefficient of 2 at 280 nm, the absorption maximum
of tryptophan, is minimal, while the emission of tryptophan, centered
around 350 nm (ΦF ) 0.12), overlaps well with the absorption band
of 2, which emits at 440 nm (ΦF ) 0.42 ( 0.04), suggesting excellent
FRET pairing (Figure 1). The critical Fo¨rster radius (R0) was
experimentally determined to be 22 Å, a suitable value for monitoring
RNA-protein recognition events.
Figure 1. Absorption (---) and emission (s) spectra of tryptophan (blue)
and 2 (red) in water.8
Figure 2. Model RRE construct and peptides used for FRET binding and
competition experiments.
is involved in the transport of immature viral mRNAs from the
nucleus to the cytoplasm of the host cell.9-13 The specific and high-
affinity binding between the protein and RNA has been attributed
to the arginine-rich domain of Rev and Stem-loop IIB of the RRE
(Figure 2).11,12,14 While the Rev protein contains a single Trp
residue (Trp45), it is strategically embedded within the RNA binding
domain.12 Residue U66 of the RRE, neighboring the binding site,
was therefore identified as the RNA modification position.6,15,18
To incorporate the modified nucleoside into RNA oligonucle-
otides, phosphoramidite 3 was prepared (Scheme 1). 5-Amino-
To unequivocally demonstrate the utility of this ribonucleoside
and its FRET pairing with natively occurring Trp residues we have
investigated the Rev peptide and the Rev Responsive Element
(RRE), its cognate RNA target. Rev, a key HIV-1 regulatory protein,
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11896 J. AM. CHEM. SOC. 2010, 132, 11896–11897
10.1021/ja105244t  2010 American Chemical Society

C O M M U N I C A T I O N S
and unmodified RRE (KD ) (3 ( 2) × 10-8) (Figure S2).8 The
placement of 2 at U66 of the RRE is therefore nonperturbing and
enables a faithful monitoring of peptide-RNA binding.
Importantly, this FRET system can also report the displacement of
the Rev peptide by competing ligands that do not contain tryptophan
residues. The RSG peptide (Figure 2) is known to have a higher affinity
to the RRE compared to Rev.11 As RSG is titrated into a solution
containing the Rev-bound labeled RRE, the emission of fluorescent
nucleoside 2, acting as the acceptor, decreases (Figure 4B). Fitting
the decreased FRET efficiency yields an IC50 value of (2 ( 1) × 10-6
M, confirming that it is indeed a tighter binder than Rev.8
In conclusion, we have identified a fluorescent nucleoside analogue
2 that is suitable for monitoring protein-RNA interactions via Fo¨rster
resonance energy transfer with native Trp residues. To the best of our
knowledge, 2 is the first isomorphic and visibly emitting nucleoside
that can efficiently pair with tryptophan. While illustrated here for the
HIV-1 Rev and RRE, the Trp-2 FRET pair is likely to find utility in
exploring other systems due to the high abundance of Trp residues
within the RNA recognition domains of proteins.21
Figure 3. Fluorescence response as labeled RRE is titrated into Rev. Inset
shows the emission spectrum at saturation. Conditions: Rev (1.0 × 10-5
M), cacodylate buffer pH 7.0 (2.0 × 10-2 M), NaCl (1.0 × 10-1 M).
Acknowledgment. We thank the National Institutes of Health
for their generous support (GM 069773), Mary Noe´ for her
assistance with MALDI experiments, and the National Science
Foundation (instrumentation grant CHE-0741968). We also thank
Prof. Akif Tezcan for the use of his fluorescence spectrometer.
Supporting Information Available: Synthetic details, thermal
denaturation measurements, titration spectra, photophysical data, and
MALDI-TOF MS spectrum. This information is available free of charge
via the Internet at http://pubs.acs.org.
Figure 4. Normalized response of the fluorescent acceptor (9) in the labeled
RRE in the following experiments: (A) titration of the labeled RRE into
Rev; (B) displacement of Rev from the labeled RRE by RSG. Conditions
same as those for Figure 3.
References
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where U66 is replaced by 2 (Figure 2). The oligonucleotide was
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(Figure S1). The folded RNA construct was as stable as the
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unnatural nucleosides. The emission profile of the emissive RRE
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the acceptor emission at 440 nm (Figure 3). At equimolar
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2 and Trp are comparable (Figure 3 inset). Based on FRET
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estimated distance between U66 and Trp.18
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emission of acceptor 2, yield a KD value of (7 ( 5) × 10-8 M, in
agreement with literature values (Figure 4A).12,16b,19 To ensure that
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anisotropy measurments20 were used to independently determine
(19) Zhang, C.-y.; Johnson, L. W. J. Am. Chem. Soc. 2006, 128, 5324–5325.
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(21) Site-directed mutagenesis can be used to engineer Trp into the RNA binding
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the affinity of Rev to both the modified (KD ) (2 ( 1) × 10-8
)
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