A highly emissive fluorescent nucleoside that signals the activity of toxic ribosome-inactivating proteins

Article abstract of DOI:10.1002/anie.200802199

Lethal weapon: The presence of RNA abasic sites generated by ribosome-inactivating proteins (RIPs) such as ricin can be detected. RNA depurination by toxins is signaled by the enhanced emission intensity of synthetically modified emissive RNA constructs that are complementary to the α-sarcin/ricin loop (see picture). This method is more efficient and precise than the use of radiolabeled substrates. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200802199

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DOI: 10.1002/anie.200802199
Fluorescent Probes
A Highly Emissive Fluorescent Nucleoside that Signals the Activity of
Toxic Ribosome-Inactivating Proteins**
Seergazhi G. Srivatsan, Nicholas J. Greco, and Yitzhak Tor*
In memory of Tuvia Bercovici
Protein toxins are among the oldest and most exotic warfare
agents.[1] Among these, ribosome-inactivating proteins
(RIPs), particularly ricin, represent some of the most poison-
ous toxins known.[2,3] Ricin is a heterodimeric protein known
to be toxic by inhalation and oral and intravenous ingestion.
Although it is less lethal than the botulinum toxin, its
accessibility and relatively simple isolation from castor
beans mean it has potential to become a weapon of choice
for bioterrorism.[4] The catastrophic impact of RIPs on
eukaryotic cells results from their action on the ribosome,
the machinery for protein biosynthesis. Ricin and saporin
catalyze the depurination of a specific nucleotide residue on a
conserved purine-rich ribosomal RNA sequence known as the
a-sarcin/ricin loop (Figure 1).[2,5,6] This N-deglycosylation
reaction reduces the affinity of the ribosome for elongation
factors that are critical for protein synthesis, thereby resulting
in disrupted protein production and ultimately cell death.[7]
The simple isolation of RIPs from natural sources has
alerted the community to the lack of useful field-operable
detection tools, as well as the absence of effective antidotes or
therapeutics for these proteins. Current approaches to RIP
detection rely on polyclonal or monoclonal antibodies that
are employed in enzyme-linked immunoadsorbent assays
(ELISA).[8,9] These tools are not easily translated to portable
devices, and cannot be straightforwardly used for the discov-
ery of inhibitors. We hypothesized that an effective approach,
amenable to high-throughput screening and possibly sensor
development, could rely on detecting the specific depurinat-
ing activity associated with RIPs, which leads to the formation
Figure 1. Ribosome inactivating protein mediated depurination of the
a-sarcin/ricin hairpin RNA substrate 1 at position A15 (corresponding
to A4324 of rat 28S rRNA) yields an RNA product 2a containing an
abasic site. Highlighted residues 9–20 represent the longest conserved
RNA sequence in the large ribosomal subunit.[6] RNA 2b represents a
model depurinated RNA product that contains a stable THF residue as
an abasic site mimic. Also shown are the chemically synthesized
of RNA abasic sites regardless of the identity of the protein.
While the generation of abasic sites in DNA is well
documented,[10,11] little is known about their formation,
significance, and repair in RNA.[12] Their occurrence appears
rather uncommon in cellular RNAs and is almost exclusively
associated with the action of RIPs. To advance an effective
method to detect RNA depurination by these toxic proteins,
we rely on: a)short oligonucleotides comprising the a-sarcin/
fluorescent probes 3–6, which contain the emissive nucleobase
analogue 7, used in this study.
ricin loop that provide authentic substrates for in vitro
applications (Figure 1),[13] and b)a new isomorphic respon-
sive fluorescent nucleoside analogue that signals changes in
its microenvironment, particularly the presence of abasic sites
(Figure 1). Here we report the use of synthetically modified
emissive RNA constructs, complementary to the a-sarcin/
ricin loop, that signal the presence of abasic RNA sites by
enhanced emission intensity upon hybridization to the
depurinated RNA product. We demonstrate the utility of
such oligonucleotides to effectively follow the enzymatic
activity of RIPs through the use of common fluorescence
spectrometric techniques.
[*] Dr. S. G. Srivatsan, Dr. N. J. Greco, Prof. Y. Tor
Department of Chemistry and Biochemistry
University of California San Diego
La Jolla, CA 92093-0358 (USA)
Fax: (+1)858-534-0202
E-mail: ytor@ucsd.edu
As part of our research into the development of fluo-
rescent nucleosides that signal the presence of nucleic acid
lesions,[11j] we found that 7 (Scheme 1), a highly emissive
analogue based on a thieno[3,4-d]pyrimidine core (fF =
[**] We are grateful to the National Institutes of Health (grant number
GM069773) for support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200802199.
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Scheme 1. Synthesis of phosphoramidite 10 for solid-phase RNA syn-
thesis. Reagents and conditions: a) DMTrCl, pyridine, RT, 62%;
b) 1. nBu2SnCl2, iPr2NEt, DCE, RT; 2. TOMCl, 808C; 3. NaHCO3, 39%;
c) iPr2NEt, CH2Cl2, iPr2NP(Cl)OEtCN, RT, 67%. TOM=CH2OSi(iPr)3;
DMTr=4,4’-dimethoxytrityl; DCE=1,2-dichloroethane.[18] See Ref. [14]
for the synthesis of 7.
Figure 2. Emission spectra of duplexes obtained upon hybridization of
RNA 11[14] to its perfect RNA and DNA complements 12 and 14,
respectively, as well as to the corresponding constructs 13 and 15
containing abasic sites. While both the perfect homo- and hetero-
duplexes (11·12 and 11·14, respectively) are highly quenched, the
duplexes containing a THF residue (a stable abasic-site analogue) are
highly emissive, with the RNA·RNA duplex 11·13 showing the highest
intensity. Conditions: duplex (1 mm) in cacodylate buffer (20 mm,
pH 7.0), NaCl (500 mm), EDTA (0.5 mm), 258C, lex =304 nm.
EDTA=ethylenediaminetetraacetic acid.[18]
0.48,[14] see Figure S1 and Table S1 in the Supporting Infor-
mation), signals the presence of abasic RNA sites with
significantly enhanced emission. Model studies showed that
the short emissive oligonucleotide 11 displayed significant
emission quenching upon hybridization to perfect comple-
ments, as well as substantial fluorescence enhancement upon
hybridization to complementary RNA and DNA oligonu-
cleotides that contain an abasic site opposite the reporter
nucleobase (Figure 2). Notably, the probe reports the pres-
ence of an abasic site in an RNA–RNA duplex with higher
signal enhancement than the corresponding RNA–DNA
duplex (Figure 2).[15] This key observation inspired the devel-
opment of the approach reported here for monitoring the
depurination activity of RIPs using fluorescent RNA probes.
Four complementary oligoribonucleotides (3–6)that
target the conserved a-sarcin/ricin stem–loop domain of
ribosomal RNA (rRNA)and place the fluorescent ribonu-
hybridized to the hairpin RNAs 1 and 2b. RNA 2b serves as a
stable model of the actual depurination reaction product 2a,
containing a tetrahydrofuran spacer as a surrogate for a d-
ribose residue that would be generated upon N-deglycosyla-
tion by RIP toxins (Figure 1). When excited at 304 nm, a
strong emission at 408 nm is observed for all single-stranded
probes 3–6 (Figure 3).[18] Significant quenching is observed
upon hybridization to the RIPs substrate 1, with the greatest
cleoside (7)opposite the common depurination site at A
15
were initially designed (Figure 1).[16] Oligonucleotide 3 com-
plements the longest universally conserved sequence (nucleo-
tides 9–20),[16] 4 targets the 3’ end of the loop (12–23), 5
targets the loop region (7–23), and 6 matches the entire stem–
loop region (Figure 1). To facilitate the site-specific incorpo-
ration of the emissive nucleoside into these singly labeled
oligonucleotides 2’-O-TOM-protected phosphoramidite 10
was synthesized and employed in standard solid-phase-syn-
thesis protocols (Scheme 1).[17,18] The integrity of the full-
length RNAs and the presence of the fluorescent nucleoside
were confirmed by MALDI mass spectrometry (see Fig-
ures S2–S5 in the Supporting Information).[18]
Figure 3. Relative emission intensities of RNA probes 3–6 (red), and
when hybridized to RNA 1 (blue) and RNA 2b (green) in cacodylate
buffer (20 mm, pH 7.0), NaCl (100 mm), EDTA (0.5 mm), 258C,
lex =304 nm, lem =408 nm.[18]
To evaluate the effectiveness of the oligonucleotides 3–6
in detecting the presence of abasic sites they were first
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quenching seen for 1·6.[19] Rewardingly, when probes 3–6 are
hybridized to 2b, considerable emission enhancements of 3.5,
1.3, 4.7, and 6.5 times respectively are observed compared to
the corresponding perfect duplexes (Figure 3). Thorough
biophysical analyses, including thermal denaturation and gel
shift experiments,[18] have indicated that oligonucleotide 6,
which encompasses the entire stem–loop structure, displayed
complete hybridization to both the RIP substrate 1 and its
depurinated analogue 2b (see Table S2 and Figure S6 in the
Supporting Information). This is in contrast to oligonucleo-
tides 3, 4, and 5, which showed complex hybridization events
and multiple product formation.[20] Taken together, these
results clearly indicate that the fluorescence enhancement
observed for the depurinated duplex 2b·6 over the perfect
duplex 1·6 arises from differences in the microenvironment of
the fluorescent nucleosides. The emissive oligonucleotide 6
was then selected to probe the enzymatic activity of RIPs.
We selected saporin as a representative toxin to inves-
tigate the N-glycosidase activity of RIPs, since it exhibits
potent depurination activity on synthetic RNAs that contain
the a-sarcin/ricin rRNA domain.[21] To identify experimental
conditions for fluorescence-based monitoring of toxin-gen-
erated RNA abasic sites, saporin-catalyzed depurination
reactions were first analyzed by polyacrylamide gel electro-
phoresis (PAGE). A solution of RIP substrate 1, spiked with
5’-end-radiolabeled 1, was thermally refolded and incubated
with saporin in tris(hydroxymethyl)aminomethane (Tris)
buffer (30 mm, pH 6.0)at 37 8C.[18] Small aliquots of the
reaction mixture were taken at short time intervals, quenched,
treated with aniline acetate at pH 4.5 (to induce strand
cleavage at abasic sites)and resolved by PAGE (Figure 4.)
The toxin-mediated reaction followed time-dependent satu-
ration kinetics, with the consumption of the substrate RNA 1
and concomitant formation of spliced products (Figure 4,
lanes 3–9). Control experiments (lanes 2 and 10) indicate that
the enzymatically generated cleaved products are exclusively
associated with the formation of abasic sites, which are known
to be susceptible to strand scission in the presence of aniline
acetate.[22] Sequencing illustrates that saporin activity largely
results in the depurination of A15 to predominantly give RNA
2a, thus suggesting that probe 6 would correctly place the
reporter nucleoside opposite the main enzymatically gener-
ated deglycosylated position (Figure 4).[23]
Figure 4. Saporin-catalyzed depurination of RNA 1 (containing trace
amounts of 5’-32P-radiolabeled 1) in Tris buffer (30 mm, pH 6.0), NaCl
(25 mm), MgCl2 (2 mm), 378C. Lanes 1 and 2: RNA substrate 1 with
and without aniline acetate treatment, respectively; lanes 3–9: reaction
mixtures sampled between 5 and 75 min and treated with aniline
acetate; lane 10: reaction duration 75 min without aniline treatment;
lanes T1 and Al, RNase T1 and alkaline hydrolysis ladder, respectively.
Strand scission with aniline gives rise to two closely migrating
products for each of the generated abasic sites, most likely from the
generation of the b-eliminated aldehyde and its aniline adduct.[12f,18]
To test the ability of RNA 6 to monitor RIP-mediated
depurination, reactions were set up with unlabeled RNA
substrate 1 and saporin as described above. At given time
intervals, aliquots were removed and immediately quenched
by adding complementary RNA 6 followed by rapid thermal
denaturation (908C)and flash-cooling renaturation (0 8C).
The emission spectrum of each sample was then recorded
(Figure 5).[18] A time-dependent fluorescence increase is
clearly observed, which reaches a plateau after 30 minutes.
The emissive ribonucleoside 7, when placed opposite a
developing abasic site, signals the depurinating activity of
saporin with an approximately sixfold overall fluorescence
enhancement.[24]
Figure 5. Saporin-catalyzed depurination reaction monitored by fluo-
rescence as a function of time. At regular time intervals, aliquots of
the toxin-mediated depurination reaction of RNA 1 were hybridized to
RNA 6 (1:0.9 ratio) in Tris buffer (30 mm, pH 6.0), NaCl (100 mm),
MgCl2 (2 mm), and emission spectra were recorded (lex =304 nm).
Inset: A plot of intensity at 410 nm versus time depicting saturation
kinetics from the formation of the highly emissive 2a·6.[18]
To confirm that the fluorescence-monitored data repre-
sents the same enzymatic transformation directly detected
using the radiolabeled RNA substrate, the two data sets were
normalized and compared to one another (Figure 6). Appar-
ent rate constants (kap)were determined by fitting the two
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Figure 6. Kinetic profiles of the saporin-mediated depurination reac-
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curves to an exponential rate equation, which gave (2.1 Æ
0.3 ” 10À3) sÀ1 and (1.7 Æ 0.2 ” 10À3) sÀ1 for the fluorescence-
and PAGE-determined depurination reactions, respec-
tively.[25] Despite the distinctly different nature of the two
techniques, excellent agreement between the two kinetic
profiles and parameters is observed. This correlation clearly
indicates that the indirect fluorescence-based sensing of
saporin activity detects the same molecular event of RNA
depurination as PAGE. Importantly, the fluorescence hybrid-
ization method is precise, safer, and far less time consuming
than the use of radiolabeled substrates, and approaches a real-
time detection of depurination activity.
In summary, we have developed a simple hybridization
assay that can detect the enzymatic activity of toxic ribosome-
inactivating proteins. It utilizes an emissive RNA oligonucle-
otide, complementary to the highly conserved a-sarcin/ricin
loop in ribosomal RNA, that contains a new fluorescent
nucleobase analogue capable of signaling the presence of
abasic RNA sites with considerably enhanced emission.
Toxin-mediated RNA depurination is, therefore, directly
translated into an increased emission signal in the visible
range (410 nm). These results demonstrate the utility of new
emissive nucleobase analogues that sense specific nucleic acid
lesions. The approach reported here could be extended to the
detection of other ribosome-inactivating proteins that act on
specific RNA substrates, and could potentially be used for the
discovery of RIP inhibitors and antidotes.
Received: May 11, 2008
Keywords: emissive nucleosides · fluorescent probes ·
.
nucleic acids · protein toxins · RNA
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monitored hybridization experiments.
[24] Importantly, saporin did not have any apparent effect on the
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6 under the above conditions (see
Figure S8 in the Supporting Information).
[25] Although the reaction was not performed under strictly pseudo
first-order conditions,
a molar ratio of 2:1 (saporin/RNA
[18] See Supporting Information for experimental details.
[19] Similar quenching effects in perfect duplexes have been
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