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A High-Throughput Fluorescence Screen to Monitor the Specific Binding of Antagonists to RNA Targets
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
261, 183–190 (1998)
AB982740
A High-Throughput Fluorescence Screen to Monitor the Specific Binding of Antagonists to RNA Targets Keita Hamasaki and Robert R. Rando1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115
Received January 9, 1998
Since RNA molecules can form intricate three-dimensional structures, it should be possible to design specific, high-affinity antagonists directed against these structures. To begin to explore the validity of this possibility, highthroughput screening methods are required to assay for RNA antagonists. A fluorescence quenching technique is described here in a 96-well plate format which is capable of screening chemical diversity libraries. A pyrene-containing aminoglycoside analog is used to accurately monitor antagonist binding to a prokaryotic 16S rRNA A-site decoding region construct. This rRNA region comprises the natural target for aminoglycoside antibiotics. The fluorescence technique reported here should be generally adaptable to monitor the binding of structurally novel antagonists to any selected RNA target. © 1998 Academic Press
RNA is an important target for aminoglycoside antibiotics (1). Aminoglycosides bind to the A-site of the decoding region in prokaryotic 16S rRNA, causing message misreading and premature polypeptide chain termination (2–5). Prokaryotic 16S rRNA is clearly too complex to study mechanistically as an aminoglycoside receptor. Fortunately, simplified 16S rRNA decoding region constructs can be fashioned which appear to behave in a biochemically expected way (6). Furthermore, the structure of a truncated 27-nt A-site decoding region construct bound to an aminoglycoside has been determined by NMR spectrometry (7). A more complex decoding region construct (129 nt) has been quantitatively studied with respect to aminoglycoside binding by fluorescence anisotropy methods (8). These studies demonstrate that aminoglycosides known to interact with the decoding region of the 16S 1 To whom correspondence should be addressed. Fax: 617-4320471.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
rRNA construct bind stoichiometrically and with dissociation constants in the 0.1–1 mM range (8). Aminoglycosides known not to bind to the decoding region did not bind to the construct (8). Experiments on simplified decoding region constructs are encouraging because they suggest that complicated RNA molecules can be deconstructed into simple units while still retaining biological activities. As indicated above, aminoglycosides bind to their rRNA target with only moderate affinities. In the dose range that these molecules are active as antibiotics, they are also fairly toxic, with this toxicity being manifest as ototoxicity and nephrotoxicity (9, 10). Also, aminoglycosides are readily susceptible to resistance, which occurs as a consequence of enzymatic modification via acylation, phosphorylation, and ADP-ribosylation mechanisms (1). Consequently, it would be highly desirable to discover molecules which had substantially higher affinities than the aminoglycosides for the decoding region (or any other suitable RNA target for that matter). It would also be important for these molecules to be less prone to metabolic inactivation than are the standard aminoglycosides. However, the design of specific inhibitors for RNA molecules is a nascent field of study. Clues to the nature of structural entities likely to bind specifically to a particular RNA molecule are scant. Simply, the rules that govern RNA–ligand recognition are not yet well understood. To these ends, it is of interest to develop highthroughput screening assays which would allow one to sample various structural types as RNA antagonists. Since aminoglycosides have been shown to not only bind to the decoding region (11), but also to the HIV2–RRE 2 Abbreviations used: HIV, human immunodeficency virus; PCP, pyrenecarbonyl-labeled paromomycin; DCC, dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; PAP, pyreneacetyl-labeled paromomycin; PBP, pyrenebutanoyl-labeled paromomycin; PCT, pyrenecarbonyl-labeled tobramycin; RRE, rev responsive element.
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transcriptional activator region (12) and to group I selfsplicing introns (13), the possibility exists for the broad pharmacological application of novel molecules with aminoglycoside-like binding characteristics. Fluorescence assays serve as ideal starting points for the design of high-throughput binding assays because they are highly sensitive, they are rapidly and inexpensively carried out in a 96-well or greater format, and these assays do not generate radioactive waste products. As mentioned above, we had previously described fluorescence-based assays to quantitate aminoglycoside–RNA binding (8, 14, 15). These assays were carried out using fluorescence anisotropy (8) or quenching assays (14). Pyrene-containing aminoglycoside analogs are useful in quenching assays because when bound to RNA molecules, the pyrene fluorescence is quenched (14). The applicability of the pyrene-based quenching assay is explored here with respect to aminoglycoside binding to a prokaryotic rRNA decoding region construct. Using a novel pyrene-labeled aminoglycoside, we have been able to adapt the fluorescence assay to a 96-well plate reader and successfully screen certain aminoglycosides. This assay will allow for the eventual screening of chemical diversity libraries aimed at the decoding region. Preliminary experiments on an HIV–RRE construct known to specifically bind aminoglycosides (8) suggest that it should also be possible to readily adapt the fluorescence plate reader assay to the screening of new analogs which bind to this region as well. MATERIALS AND METHODS
Materials Paromomycin, kanamycin B, tobramycin, and hygromycin B were from Sigma. Neomycin, gentamycin, and streptomycin were purchased from Fluka. 1-Pyrenebutanoic acid succiimidyl ester was purchased from Molecular Probes. Pyreneacetic acid and pyrenecarboxylic acid were purchased from Aldrich. Template DNA and primers for 16S rRNA analog were from Oligo.etc. Template DNA and primers for RRE IIB were from Integrated DNA Technology. PCR reactions were carried out using Taq DNA polymerase from Promega. RNA transcripts were generated using the RiboMAX large-scale RNA production kit with T7 RNA polymerase from Promega. Sephadex G-50 was from Pharmacia. All of the syntheses described below were carried out by the previously published procedure (8). Pyrenecarbonyl-labeled paromomycin (PCP). Pyrenecarbonyl-labeled paromomycin (PCP), pyrenecarboxylic acid (26 mg, ca. 100 mmol), and dicyclohexylcarbodiimide (DCC; 21 mg, ca. 100 mmol) were dissolved in N,N,dimethylformamide (DMF) and stirred at ;5°C for 1 h. N-Hydroxysucciimide (12 mg, 100 mmol) was added to above solution and stirred for 1 h. The reaction mixture
was filtered to remove precipitate. This solution was then added to the DMF (3 mL) and an aqueous solution (1.5 mL) of paromomycin (90 mg, ca 150 mmol), and stirred for 10 h. The solvent was removed by evaporation. The product was partially purified by Sephadex CM-25 ion-exchange column chromatography and then completely purified on an ODS column (Rainin 5 3 26 mm) by HPLC (Waters 625 LC system) with a of acetonitrile and water gradient containing 0.1% trifluoroacetic acid (TFA). 1H NMR in D2O, 500 MHz d 5 1.7756 (t, 1H), 2.4570 (m, 1H), 2.9467 (q, 1H), 3.1011 (t, 1H), 3.2868–3.9458 (m), 4.1980 (q, 1H), 4.3341 (broad, s, 1H), 4.3757 (d, 1H), 4.4782 (t, 1H), 5.2815 (d, 1H), 4.5335 (d, 1H), 4.4782 (t, 1H), 4.5335 (d, 1H), 5.2815 (d, 1H), 5.4004 (s, 1H), 8.2389 – 8.4774 (m, 9H). FABMS (M1): 844. Pyreneacetyl-labeled paromomycin (PAP). PAP was synthesized according to the same procedure described above for PCP, save for the substitution of pyreneacetic acid for pyrenecarboxylic acid. 1H NMR in D2O, 500 MHz d 5 1.8425 (q, 1H), 2.1904 (q, 1H), 2.5010 (m, 1H), 2.8441 (q, 1H), 2.9355 (t, 1H), 3.1862 (t, 1H), 3.2321 (q, 1H), 3.4069 – 4.0906 (m), 4.3377 (d, 1H), 4.4503 (d,1H), 4.9114 (s,1H), 5.0874 (d,1H), 5.6343 (d,1H), 8.0762– 8.4109 (m, 9H). FABMS (M1): 858. Pyrenebutanoyl-labeled paromomycin (PBP). Pyrenebutanoic acid succiimidyl ester (40 mg, ca. mmol) and paromomycin (90 mg, ca. 150 mmol) were dissolved in a DMF-aqueous solution and stirred for 10 h. The solvent was removed by evaporation. The product was partially purified by Sephadex CM-25 ion-exchange column chromatography and then purified on an ODS column (Rainin 5 3 26 mm), by HPLC (Waters 625 LC system) with a gradient of acetonitrile and water. The solvents contained 0.1% of trifluoroacetic acid. The product was partially purified by Sephadex CM-25 ionexchange column chromatography and completely purified on an ODS column (Rainin 5 3 26 mm) by HPLC (Waters 625 LC system) using a gradient of acetonitrile and H2O in the presence of 0.1% trifluoroacetic acid. 1H NMR in D2O, 500 MHz d 5 1.8407 (q, 4H), 2.5355 (m, 2H), 3.4071–3.9873 (m, Majority), 4.3342 (d, 3H), 4.4783 (t, 1H), 5.4045 (s, 1H), 5.7336 (d, 2H), 8.0929 – 8.5449 (m, 9H). FABMAS (M1H1): 886. Pyrenecarbonyl-labeled tobramycin (PCT). PCT was prepared by exactly the same procedure as that described for PCP. 1H NMR in D2O, 500 MHz d 5 1.888 (q, 1H), 2.1402 (q, 1H), 2.4335 (d, 1H), 2.5546 (d, 1H), 3.4874 (t, 1H), 3.6112– 4.1593 (m), 4.9764 (s, 1H), 5.7046 (d, 1H), 8.2165– 8.4520 (m, 9H). FABMS (M1): 696. Methods Fluorescence measurements. Pyrene-labeled paromomycin (PBP, PAP, PCP, and PCT) concentrations
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were determined spectroscopically at 340 nm using a molar extinction coefficient of 4.2 3 104 M21 cm21. Fluorescence measurements were performed on a Perkin–Elmer LS-50B luminescence spectrometer equipped with either a standard cuvette holder or a 96-well plate reader. The tracer solution was excited at 340 nm and monitored at 380 nm. Measurements were performed in a buffer solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 20 mM Hepes (pH 7.5). All measurements, save those reported in Fig. 7, were carried out in cuvettes. The experiments reported in Fig. 7 were carried out in a plate reader. Determination of dissociation constants. The dissociation constant between the RNA (R) and the tracer (T) is defined as
(Kd) (Fig. 3– 6A). Calculations were performed using Kaleidagraph. The affinity of the analog RNA for the aminoglycosides is determined by competition between the aminoglycosides and the tracer. The dissociation constants between the RNA and the aminoglycoside (KD) are defined by RNA z aminoglycoside KºD RNA 1 aminoglycoside [8] K D 5 @RNA] [aminoglycoside]/ [RNAzaminoglycoside]
[9]
RNA z T Kºd RNA 1 T
[1]
The initial concentrations of the RNA and the aminoglycoside are defined by
K d 5 @RNA] [T] / [RNAzT]
[2]
[RNA]0 5 [RNAzaminoglycoside]
The initial concentrations, [RNA]0 and [T]0 are defined by [RNA]0 5 [RNA] 1 [RNAzT]
[3]
[T]0 5 [T] 1 [RNAzT]
[4]
The fluorescence signal value of the sample solution is defined by i 5 i 0 1 Di @RNAzT],
[5]
where i and i0 are the fluorescence signal (anisotropy or intensity) of the tracer in the presence and in the absence of RNA, respectively. Di is the difference in the fluorescence signal of 1 nM of the tracer in the presence of an infinite concentration of RNA and in the absence of RNA. Equations [2], [3], and [4] give RNA z T complex concentrations as [RNAzT] 5 ([RNA]0 1 [T]0 1 K d 2 ~~@RNA]0 1 [T]0 1 K d! 2 2 4 @RNA]0 [T]0)1/2)/2
[6]
[10]
[Aminoglycoside]0 5 [aminoglycoside] 1 [RNAzaminoglycoside]
[11]
The concentrations of the other species are denoted by [RNAzT] 5 [T]0 ~i 2 i 0! / ~i ` 2 i 0!
[12]
[T] 5 [T]0 (12~i 2 i 0! / ~i ` 2 i 0!)
[13]
The fluorescence signal values of the totally bound tracer are estimated by Eq. [7] and i ` 5 i 1 Di [T0] / nM
[14]
Then, the following equation is readily derived from the equations shown above. [Aminoglycoside]0 5 ~K D ~i ` 2 i! / K d ~i 2 i 0! 1 1! 3 ~@RNA]0 2 K d ~i 2 i 0!/~i ` 2 i!
Then the following equation is obtained from Eqs. [5] and [6]: i 5 i 0 1 Di ~~@RNA]0 1 [T]0 1 K d 2 ~~@RNA]0 1 [T]0 1 K d! 2 2 4 @RNA]0 [T]0)1/2)/2
1 [RNAzT] 1 [RNA]
[7]
Equation [7] was used for the determination of the dissociation constant between the RNA and the tracer
2 @T]0 ~i 2 i 0! / ~i ` 2 i 0!!
[15]
In the competitive binding assay, Eq. [15] is used for the calculation of KD, the dissociation constant for the RNA and aminoglycosides interactions (Figs. 3– 6B). Both Kd and KD were determined by nonlinear curvefitting by the corresponding equations described above (8). Calculations were performed using Kaleidagraph.
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ety and the RNA, then full recovery is expected occur. Figures 2A–2C show the fluorescence emission spectra of PBP, PAP, and PCP solutions (10 nM) containing 1 mM of RNA 16S rRNA analog alone and in the presence of various concentrations of paromomycin. The fluorescence intensity of all of the pyrene-labeled paromomycin analogs increased upon the addition of unlabeled paromomycin. These results show that competitive binding of fluorescently labeled and unlabeled paromomycin occurs with the 16S rRNA analog. The dissociation constants between paromomycin and the 16S rRNA analog were determined by competitive titration (8). The binding results are summarized on Table 1. As expected, all of the measured dissociation constants for paromomycin are close to each other, demonstrating
SCHEME 1.
Tracers used in drug screening and binding assays.
RESULTS
The Binding of Fluorescent Probes to the Decoding Region and HIV–RRE RNA Constructs Initial experiments were performed by studying the RNA-induced quenching behavior of three candidate pyrene containing paromomycin analogs shown in Scheme 1 (PBP, PAP, and PCP). Figures 1A–1C show the fluorescence emission spectra of 10 nM solutions of PBP, PAP, and PCP, respectively, alone and in the presence of various concentrations of our previously reported 109-nt 16S rRNA decoding region analog (8). Upon the addition of the RNA, the fluorescence emission intensity of all three pyrene-labeled paromomycin analogs decreased markedly. Moreover, each data point precisely fits the curve obtained from an equation derived assuming 1:1 binding stoichiometry (8). The titration results provide dissociation constants for PBP, PAP, and PCP of 235 6 34, 301 6 13, and 263 6 13 nM, respectively. The studies described above show that PBP, PAP, and PCP show appropriate quenching behavior when treated with the RNA construct. In the next series of experiments, competition assays were set up between the three analogs and paromomycin, an aminoglycoside known to bind to the A-site of the decoding region (7). These competition assays are essential for the success of any high-throughput screen based on the quenching assays. An important issue to address here is whether complete fluorescence recovery occurs when competing aminoglycosides are added. If nonactive site interactions do not occur between the fluorescent moi-
FIG. 1. (A, B, and C) Fluorescence spectra of PBP, PAP, and PCP solutions (10 nM) alone (1) and in the presence of 100 nM (2), 200 nM (3), 300 nM (4), 400 nM (5), 500 nM (6), 600 nM (7), 700 nM (8), 800 nM (9), 900 nM (10), and 1000 nM (11) of RNA.
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cin tested (2 mM). Using PAP (Fig. 4), approximately 90% recovery is observed. On the other hand, the fluorescence intensity of PCP completely recovered to its initial value in the presence of high concentrations of paromomycin, as shown in Fig. 5. Therefore, PCP shows no measurable nonactive site binding due to the pyrene moiety and was thus chosen as the most promising tracer for use in the screening of molecules targeted against the 16S rRNA decoding region. Previously we showed that tobramycin binds to the an HIV–RRE IIB construct of the RRE transcriptional activator region (8). It was of interest to determine if PCT (Scheme 1) would be spectroscopically as wellbehaved in this system as PCP is in the decoding region construct. As shown in Fig. 6A, PCT can be titrated using the RRE construct, providing a dissociation constant of 0.114 6 0.006 mM for the probe. Importantly, when PCT–RRE complexes were titrated with tobramycin, complete recovery of fluorescence was observed (Fig. 6B) and a dissociation constant of 1.56 6 0.62 mM could be calculated for tobramycin. Therefore, PCT shows precisely the kind of fluorescent spectroscopic behavior required of an analog to be used in competitive screening assays. Drug Screening Using the Fluorescence Plate Reader The next step to take in establishing a high throughput fluorescence assay involves adapting the quenching assay described using PCP to a 96-well plate format A solution was prepared containing 500 nM of PCP and 1 mM of the 16S rRNA construct in buffer. Each well of the plate contained 200 mL of above solution and 100 mM of an aminoglycoside aliquot to be tested. The fluorescence emission intensity of PCP, in the well containing 1 mM of the 16S rRNA analog, recovered to FIG. 2. (A, B, and C) Fluorescence spectra of PBP, PAP, and PCP solutions (10 nM) containing 1 mM of RNA and in the absence (1) and in the presence of 10 mM (2), 20 mM (3), 30 mM (4), 40 mM (5), 50 mM (6), 60 mM (7), 70 mM (8), 80 mM (9), 90 mM (10), and 100 mM (11) of paromomycin.
TABLE 1
Dissociation Constants for the Binding of Aminoglycosides to the 16S rRNA Construct i
internal consistency. Furthermore, the dissociation constants measured for paromomycin using these pyrene derivatives are also close to one obtained by a fluorescence anisotropy assay using a different probe (CRP, Scheme 1) (15). While the dissociation constants obtained using PBP and PAP are close to the value obtained using CRP, the fluorescence intensities using the pyrene analogs did not completely recover to their initial values in the presence of high concentrations (2 mM) of paromomycin, suggesting some possible nonspecific binding of the pyrene moiety to RNA in these instances. In the case of PBP (Fig. 3), approximately 90% fluorescence recovery is obtained at the highest concentration of paromomy-
[RNA] 5 1 mM o
i[paromomycin] 5 2 mM/io
Kd (mM)
KD (mM)
0.39 0.31 0.35 —
1 0.9 0.9 —
0.235 6 0.034 0.301 6 0.013 0.263 6 0.013 0.165 6 0.031
2.14 6 0.57 1.29 6 0.21 2.65 6 0.88 1.85 6 0.32
/i
PCP PAP PBP CRPa
Note. io, fluorescence intensity of pyrene-labled paromomycin solution (10 nM) alone. i[RNA] 5 1 mM, fluorescence intensity of pyrenelabled paromomycin solution (10 nM) in the presence of 1 mM of the 16s rRNA analog. i[paromomycin] 5 2 mM, fluorescence intensity of pyrene-labled paromomycin (10 nM) containing 1 mM of the 16s rRNA analog, in the presence of 2 mM of paromomycin. Kd, dissociation constants between the pyrene-labeled paromomycin and the 16S rRNA analog. KD, dissociation constants between paromomycin and the 16S rRNA analog obtained by each tracer. a Previously published in Ref. (8).
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diethylethylglucosamine, and N-heptyl N-hexyl-paraaminoaniline, which should not bind to the decoding region construct, do not compete with PCP as revealed by this assay. DISCUSSION
Since RNA molecules can form intricate three-dimensional structures, it ought to be possible to design small organic molecules to bind to functional domains of RNA. Aminoglycoside antibiotics serve as starting points for design because they have been shown to have a general affinity for RNA molecules (16 –20). With a few notable exceptions (15, 20), the affinity of aminoglycosides for RNA molecules is in the micromole range (16 –19). As more is understood concerning the nature of the specific interactions between aminoglycosides and target RNA species, it should be possible to design higher affinity analogs.
FIG. 3. (A) Relative fluorescence intensity of PBP (10 nM) solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PBP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
varying degrees in the presence of 100 mM of the selected aminoglycosides. Figure 7 show the results of the fluorescence recovery after the addition of the aminoglycosides. In the presence of 100 mM of neomycin, the fluorescence intensity of PCP recovered to 60% of its original intensity. In the presence of paromomycin, kanamycin B, gentamycin, and tobramycin only 30 to 40% fluorescence recovery occurred. By contrast, the fluorescence recovery of PCP caused by the addition of hygromycin B and streptomycin proved to be negligible. These results are consistent with our previous results. The 16S rRNA decoding region analog binds neomycin 10 times tighter than the other aminoglycosides. Furthermore, the construct does not bind either hygromycin B or streptomycin as revealed by fluorescence anisotropy assays (15). In addition, many diverse amine containing molecules, including glucosamine, spermine, dopamine, 4-methoxytryptamine, 1-b-N,N-
FIG. 4. (A) Relative fluorescence intensity of PAP (10 nM), solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PAP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
FLUORESCENCE ASSAYS TO STUDY RNA ANTAGONISTS
189
in the 1 mM range (8). As expected, neither hygromycin nor streptomycin measurably bind (8). In the current work, a fluorescence-quenching assay was developed using the pyrene-labeled paromomycin analogs PBP, PAP, and PCP to monitor the binding of aminoglycosides to the decoding region construct. The fluorescence emission of all three analogs was readily quenched with the decoding region RNA, allowing direct binding constants to be calculated for the three analogs. However, only PCP showed 100% fluorescence recovery upon the addition of high concentrations of paromomycin. In addition, the dissociation constant for added paromomycin in the competition assay using PCP was measured to be 2.14 6 0.57 mM, a value within experimental error of the dissociation constant measured using a fluorescence anisotropy assay and a different fluorescent probe (8). Hence, PCP was chosen as the probe most applicable for use in the screening assay.
FIG. 5. (A) Relative fluorescence intensity of PCP (10 nM), solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PCP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
There are several naturally occurring RNA constructs of substantial interest with respect to RNA antagonist design. The prokaryotic 16S decoding region and the HIV–RRE transactivator domain are two of these targets. Simple constructs from both RNA regions bind aminoglycosides (6 – 8, 12). In the studies reported here, it was of interest to develop fluorescence-based screens for targets of this type to enable the discovery of new antagonists of biologically interesting RNA targets. The work is largely focused on the decoding region construct. It had previously been shown that the 129-nt decoding region construct under consideration here stoichiometrically binds aminoglycoside antibiotics with the expected affinities. That is, neomycin B binds with a dissociation constant of approximately 0.1 mM, and other aminoglycosides, including tobramycin, gentamycin, and paromomycin, have dissociation constants
FIG. 6. (A) Relative fluorescence intensity of PCT (10 nM) solution as a function of increasing concentrations of the RRE IIB RNA analog. (B) Relative fluorescence intensity of PCT (10 nM) solution containing 1 mM of RRE IIB RNA analog as a function of increasing concentrations of the paromomycin concentration.
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Fig. 4 that PCT would be an ideal candidate for screening studies aimed at discovering novel antagonists of the RRE region of RNA. The experiments reported here show that fluorescent ligands which bind to RNA with fluorescence quenching can be used in the design of rapid and quantitative screening assays for uncovering RNA antagonists. ACKNOWLEDGMENTS These studies were partially funded by U.S. Public Health Service National Institutes of Health Grants EY-03624 and EY-04096. K.H. was funded by a research fellowship from the Japan Society for the Promotion of Science.
REFERENCES FIG. 7. Fluorescence recovery by competitive binding of aminoglycosides. io, fluorescence intensity of PCP (500 nM). iRNA, fluorescence intensity of PCP (500 nM) containing 1 mM of RNA. i, fluorescence intensity of PCP (500 nM) containing 1 mM of RNA in the presence of 100 mM of the aminoglycosides.
PCP was added to the wells in a 96-well plate format along with the decoding region construct. Various aminoglycosides, which were already known to bind to the A-site of the decoding region with varying affinities (8), were then added and the recovery of fluorescence was determined. The results from the screening assay were exactly as predicted from previous studies in which precise determinations of dissociation constants between aminoglycosides and the decoding region construct were made (8). This demonstrates that PCP can be used to access the affinities of analogs directed against the A-site of the decoding region. The screen described here does not, of course, provide an exact dissociation constant for a particular molecule which antagonizes PCP action. Molecules which prove to have activity in this screen can then be quantitatively assayed by standard fluorescent methods (8) to determine precise dissociation constants and stoichiometry of binding. Determination of the stoichiometry of binding is especially important for accessing the specificity of action of an RNA antagonist. The 96-well plate screen is currently being used in our laboratory to uncover novel structural entities capable of competing with aminoglycoside for binding to the decoding region. While PCT was not investigated as thoroughly with respect to the HIV–RRE region as was PCP investigated as a probe for the decoding region, it is clear from
1. Chambers, H. F., and Sande, M. A. (1996) in Goodman & Gilman’s The Pharmacological Basis of Therapeutics (Hardman, J. G., Limbird, L. E., Molinoff, P. B., Ruddon, R. W., and Gilman, A. G., Eds.), ninth ed., pp. 1103–1121. 2. Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. H., and Waring, M. J. (1981) The Molecular Basis of Antibiotic Action, Wiley, New York. 3. De Stasio, E. A., Moazed, D., Noller, H. F., and Dahlberg, A. E. (1989) EMBO J. 8, 1213–1216. 4. Noller, H. F. (1991) Annu. Rev. Biochem. 60, 191–227. 5. Cundliffe, E. (1989) Annu. Rev. Microbiol. 43, 207–233. 6. Purohit, P., and Stern, S. (1994) Nature 370, 659 – 662. 7. Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367–1371. 8. Wang, Y., Hamasaki, K., and Rando, R. R. (1997) Biochemistry 36, 768 –779. 9. Hutchin, T., and Cortopassi, G. (1994) Antimicrob. Agents Chemother. 38, 2517–2520. 10. Lietman, P. S., and Smith, C. R. (1983) J. Infect. Dis. 5(Suppl. 2), S284 –S292. 11. De Stasio, E. A., and Dahlberg, A. E. (1990) J. Mol. Biol. 212, 127–133. 12. Zapp, M. L., Stern, S., and Green, M. R. (1993) Cell 74, 969 –978. 13. von Ahsen, U., Davies, J., and Schroeder, R. (1991) Nature 353, 368 –370. 14. Wang, Y., and Rando, R. R. (1995) Chem. Biol. 2, 281–290. 15. Wang, Y., Killian, J., Hamasaki, K., and Rando, R. R. (1996) Biochemistry 35, 12338 –12346. 16. Tangy, F., Capmau, M.-L., and LeGoffic, F. (1983) Eur. J. Biochem. 131, 581–587. 17. Stage, T. K., Hertel, K. J., and Uhlenbeck, O. C. (1995) RNA 1, 95–101. 18. Lato, S. M., and Ellington, A. D. (1996) Mol. Div. 2, 103–110. 19. Wallis, M. G., Von Ahsen, U., Schroeder, R., and Famulok, M. A. (1995) Chem. Biol. 2, 543–552. 20. Hamasaki, K., Killian, J., Cho, J., and Rando, R. R. (1998) Biochemistry 37, 656 – 663.
261, 183–190 (1998)
AB982740
A High-Throughput Fluorescence Screen to Monitor the Specific Binding of Antagonists to RNA Targets Keita Hamasaki and Robert R. Rando1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115
Received January 9, 1998
Since RNA molecules can form intricate three-dimensional structures, it should be possible to design specific, high-affinity antagonists directed against these structures. To begin to explore the validity of this possibility, highthroughput screening methods are required to assay for RNA antagonists. A fluorescence quenching technique is described here in a 96-well plate format which is capable of screening chemical diversity libraries. A pyrene-containing aminoglycoside analog is used to accurately monitor antagonist binding to a prokaryotic 16S rRNA A-site decoding region construct. This rRNA region comprises the natural target for aminoglycoside antibiotics. The fluorescence technique reported here should be generally adaptable to monitor the binding of structurally novel antagonists to any selected RNA target. © 1998 Academic Press
RNA is an important target for aminoglycoside antibiotics (1). Aminoglycosides bind to the A-site of the decoding region in prokaryotic 16S rRNA, causing message misreading and premature polypeptide chain termination (2–5). Prokaryotic 16S rRNA is clearly too complex to study mechanistically as an aminoglycoside receptor. Fortunately, simplified 16S rRNA decoding region constructs can be fashioned which appear to behave in a biochemically expected way (6). Furthermore, the structure of a truncated 27-nt A-site decoding region construct bound to an aminoglycoside has been determined by NMR spectrometry (7). A more complex decoding region construct (129 nt) has been quantitatively studied with respect to aminoglycoside binding by fluorescence anisotropy methods (8). These studies demonstrate that aminoglycosides known to interact with the decoding region of the 16S 1 To whom correspondence should be addressed. Fax: 617-4320471.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
rRNA construct bind stoichiometrically and with dissociation constants in the 0.1–1 mM range (8). Aminoglycosides known not to bind to the decoding region did not bind to the construct (8). Experiments on simplified decoding region constructs are encouraging because they suggest that complicated RNA molecules can be deconstructed into simple units while still retaining biological activities. As indicated above, aminoglycosides bind to their rRNA target with only moderate affinities. In the dose range that these molecules are active as antibiotics, they are also fairly toxic, with this toxicity being manifest as ototoxicity and nephrotoxicity (9, 10). Also, aminoglycosides are readily susceptible to resistance, which occurs as a consequence of enzymatic modification via acylation, phosphorylation, and ADP-ribosylation mechanisms (1). Consequently, it would be highly desirable to discover molecules which had substantially higher affinities than the aminoglycosides for the decoding region (or any other suitable RNA target for that matter). It would also be important for these molecules to be less prone to metabolic inactivation than are the standard aminoglycosides. However, the design of specific inhibitors for RNA molecules is a nascent field of study. Clues to the nature of structural entities likely to bind specifically to a particular RNA molecule are scant. Simply, the rules that govern RNA–ligand recognition are not yet well understood. To these ends, it is of interest to develop highthroughput screening assays which would allow one to sample various structural types as RNA antagonists. Since aminoglycosides have been shown to not only bind to the decoding region (11), but also to the HIV2–RRE 2 Abbreviations used: HIV, human immunodeficency virus; PCP, pyrenecarbonyl-labeled paromomycin; DCC, dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; PAP, pyreneacetyl-labeled paromomycin; PBP, pyrenebutanoyl-labeled paromomycin; PCT, pyrenecarbonyl-labeled tobramycin; RRE, rev responsive element.
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transcriptional activator region (12) and to group I selfsplicing introns (13), the possibility exists for the broad pharmacological application of novel molecules with aminoglycoside-like binding characteristics. Fluorescence assays serve as ideal starting points for the design of high-throughput binding assays because they are highly sensitive, they are rapidly and inexpensively carried out in a 96-well or greater format, and these assays do not generate radioactive waste products. As mentioned above, we had previously described fluorescence-based assays to quantitate aminoglycoside–RNA binding (8, 14, 15). These assays were carried out using fluorescence anisotropy (8) or quenching assays (14). Pyrene-containing aminoglycoside analogs are useful in quenching assays because when bound to RNA molecules, the pyrene fluorescence is quenched (14). The applicability of the pyrene-based quenching assay is explored here with respect to aminoglycoside binding to a prokaryotic rRNA decoding region construct. Using a novel pyrene-labeled aminoglycoside, we have been able to adapt the fluorescence assay to a 96-well plate reader and successfully screen certain aminoglycosides. This assay will allow for the eventual screening of chemical diversity libraries aimed at the decoding region. Preliminary experiments on an HIV–RRE construct known to specifically bind aminoglycosides (8) suggest that it should also be possible to readily adapt the fluorescence plate reader assay to the screening of new analogs which bind to this region as well. MATERIALS AND METHODS
Materials Paromomycin, kanamycin B, tobramycin, and hygromycin B were from Sigma. Neomycin, gentamycin, and streptomycin were purchased from Fluka. 1-Pyrenebutanoic acid succiimidyl ester was purchased from Molecular Probes. Pyreneacetic acid and pyrenecarboxylic acid were purchased from Aldrich. Template DNA and primers for 16S rRNA analog were from Oligo.etc. Template DNA and primers for RRE IIB were from Integrated DNA Technology. PCR reactions were carried out using Taq DNA polymerase from Promega. RNA transcripts were generated using the RiboMAX large-scale RNA production kit with T7 RNA polymerase from Promega. Sephadex G-50 was from Pharmacia. All of the syntheses described below were carried out by the previously published procedure (8). Pyrenecarbonyl-labeled paromomycin (PCP). Pyrenecarbonyl-labeled paromomycin (PCP), pyrenecarboxylic acid (26 mg, ca. 100 mmol), and dicyclohexylcarbodiimide (DCC; 21 mg, ca. 100 mmol) were dissolved in N,N,dimethylformamide (DMF) and stirred at ;5°C for 1 h. N-Hydroxysucciimide (12 mg, 100 mmol) was added to above solution and stirred for 1 h. The reaction mixture
was filtered to remove precipitate. This solution was then added to the DMF (3 mL) and an aqueous solution (1.5 mL) of paromomycin (90 mg, ca 150 mmol), and stirred for 10 h. The solvent was removed by evaporation. The product was partially purified by Sephadex CM-25 ion-exchange column chromatography and then completely purified on an ODS column (Rainin 5 3 26 mm) by HPLC (Waters 625 LC system) with a of acetonitrile and water gradient containing 0.1% trifluoroacetic acid (TFA). 1H NMR in D2O, 500 MHz d 5 1.7756 (t, 1H), 2.4570 (m, 1H), 2.9467 (q, 1H), 3.1011 (t, 1H), 3.2868–3.9458 (m), 4.1980 (q, 1H), 4.3341 (broad, s, 1H), 4.3757 (d, 1H), 4.4782 (t, 1H), 5.2815 (d, 1H), 4.5335 (d, 1H), 4.4782 (t, 1H), 4.5335 (d, 1H), 5.2815 (d, 1H), 5.4004 (s, 1H), 8.2389 – 8.4774 (m, 9H). FABMS (M1): 844. Pyreneacetyl-labeled paromomycin (PAP). PAP was synthesized according to the same procedure described above for PCP, save for the substitution of pyreneacetic acid for pyrenecarboxylic acid. 1H NMR in D2O, 500 MHz d 5 1.8425 (q, 1H), 2.1904 (q, 1H), 2.5010 (m, 1H), 2.8441 (q, 1H), 2.9355 (t, 1H), 3.1862 (t, 1H), 3.2321 (q, 1H), 3.4069 – 4.0906 (m), 4.3377 (d, 1H), 4.4503 (d,1H), 4.9114 (s,1H), 5.0874 (d,1H), 5.6343 (d,1H), 8.0762– 8.4109 (m, 9H). FABMS (M1): 858. Pyrenebutanoyl-labeled paromomycin (PBP). Pyrenebutanoic acid succiimidyl ester (40 mg, ca. mmol) and paromomycin (90 mg, ca. 150 mmol) were dissolved in a DMF-aqueous solution and stirred for 10 h. The solvent was removed by evaporation. The product was partially purified by Sephadex CM-25 ion-exchange column chromatography and then purified on an ODS column (Rainin 5 3 26 mm), by HPLC (Waters 625 LC system) with a gradient of acetonitrile and water. The solvents contained 0.1% of trifluoroacetic acid. The product was partially purified by Sephadex CM-25 ionexchange column chromatography and completely purified on an ODS column (Rainin 5 3 26 mm) by HPLC (Waters 625 LC system) using a gradient of acetonitrile and H2O in the presence of 0.1% trifluoroacetic acid. 1H NMR in D2O, 500 MHz d 5 1.8407 (q, 4H), 2.5355 (m, 2H), 3.4071–3.9873 (m, Majority), 4.3342 (d, 3H), 4.4783 (t, 1H), 5.4045 (s, 1H), 5.7336 (d, 2H), 8.0929 – 8.5449 (m, 9H). FABMAS (M1H1): 886. Pyrenecarbonyl-labeled tobramycin (PCT). PCT was prepared by exactly the same procedure as that described for PCP. 1H NMR in D2O, 500 MHz d 5 1.888 (q, 1H), 2.1402 (q, 1H), 2.4335 (d, 1H), 2.5546 (d, 1H), 3.4874 (t, 1H), 3.6112– 4.1593 (m), 4.9764 (s, 1H), 5.7046 (d, 1H), 8.2165– 8.4520 (m, 9H). FABMS (M1): 696. Methods Fluorescence measurements. Pyrene-labeled paromomycin (PBP, PAP, PCP, and PCT) concentrations
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were determined spectroscopically at 340 nm using a molar extinction coefficient of 4.2 3 104 M21 cm21. Fluorescence measurements were performed on a Perkin–Elmer LS-50B luminescence spectrometer equipped with either a standard cuvette holder or a 96-well plate reader. The tracer solution was excited at 340 nm and monitored at 380 nm. Measurements were performed in a buffer solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 20 mM Hepes (pH 7.5). All measurements, save those reported in Fig. 7, were carried out in cuvettes. The experiments reported in Fig. 7 were carried out in a plate reader. Determination of dissociation constants. The dissociation constant between the RNA (R) and the tracer (T) is defined as
(Kd) (Fig. 3– 6A). Calculations were performed using Kaleidagraph. The affinity of the analog RNA for the aminoglycosides is determined by competition between the aminoglycosides and the tracer. The dissociation constants between the RNA and the aminoglycoside (KD) are defined by RNA z aminoglycoside KºD RNA 1 aminoglycoside [8] K D 5 @RNA] [aminoglycoside]/ [RNAzaminoglycoside]
[9]
RNA z T Kºd RNA 1 T
[1]
The initial concentrations of the RNA and the aminoglycoside are defined by
K d 5 @RNA] [T] / [RNAzT]
[2]
[RNA]0 5 [RNAzaminoglycoside]
The initial concentrations, [RNA]0 and [T]0 are defined by [RNA]0 5 [RNA] 1 [RNAzT]
[3]
[T]0 5 [T] 1 [RNAzT]
[4]
The fluorescence signal value of the sample solution is defined by i 5 i 0 1 Di @RNAzT],
[5]
where i and i0 are the fluorescence signal (anisotropy or intensity) of the tracer in the presence and in the absence of RNA, respectively. Di is the difference in the fluorescence signal of 1 nM of the tracer in the presence of an infinite concentration of RNA and in the absence of RNA. Equations [2], [3], and [4] give RNA z T complex concentrations as [RNAzT] 5 ([RNA]0 1 [T]0 1 K d 2 ~~@RNA]0 1 [T]0 1 K d! 2 2 4 @RNA]0 [T]0)1/2)/2
[6]
[10]
[Aminoglycoside]0 5 [aminoglycoside] 1 [RNAzaminoglycoside]
[11]
The concentrations of the other species are denoted by [RNAzT] 5 [T]0 ~i 2 i 0! / ~i ` 2 i 0!
[12]
[T] 5 [T]0 (12~i 2 i 0! / ~i ` 2 i 0!)
[13]
The fluorescence signal values of the totally bound tracer are estimated by Eq. [7] and i ` 5 i 1 Di [T0] / nM
[14]
Then, the following equation is readily derived from the equations shown above. [Aminoglycoside]0 5 ~K D ~i ` 2 i! / K d ~i 2 i 0! 1 1! 3 ~@RNA]0 2 K d ~i 2 i 0!/~i ` 2 i!
Then the following equation is obtained from Eqs. [5] and [6]: i 5 i 0 1 Di ~~@RNA]0 1 [T]0 1 K d 2 ~~@RNA]0 1 [T]0 1 K d! 2 2 4 @RNA]0 [T]0)1/2)/2
1 [RNAzT] 1 [RNA]
[7]
Equation [7] was used for the determination of the dissociation constant between the RNA and the tracer
2 @T]0 ~i 2 i 0! / ~i ` 2 i 0!!
[15]
In the competitive binding assay, Eq. [15] is used for the calculation of KD, the dissociation constant for the RNA and aminoglycosides interactions (Figs. 3– 6B). Both Kd and KD were determined by nonlinear curvefitting by the corresponding equations described above (8). Calculations were performed using Kaleidagraph.
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ety and the RNA, then full recovery is expected occur. Figures 2A–2C show the fluorescence emission spectra of PBP, PAP, and PCP solutions (10 nM) containing 1 mM of RNA 16S rRNA analog alone and in the presence of various concentrations of paromomycin. The fluorescence intensity of all of the pyrene-labeled paromomycin analogs increased upon the addition of unlabeled paromomycin. These results show that competitive binding of fluorescently labeled and unlabeled paromomycin occurs with the 16S rRNA analog. The dissociation constants between paromomycin and the 16S rRNA analog were determined by competitive titration (8). The binding results are summarized on Table 1. As expected, all of the measured dissociation constants for paromomycin are close to each other, demonstrating
SCHEME 1.
Tracers used in drug screening and binding assays.
RESULTS
The Binding of Fluorescent Probes to the Decoding Region and HIV–RRE RNA Constructs Initial experiments were performed by studying the RNA-induced quenching behavior of three candidate pyrene containing paromomycin analogs shown in Scheme 1 (PBP, PAP, and PCP). Figures 1A–1C show the fluorescence emission spectra of 10 nM solutions of PBP, PAP, and PCP, respectively, alone and in the presence of various concentrations of our previously reported 109-nt 16S rRNA decoding region analog (8). Upon the addition of the RNA, the fluorescence emission intensity of all three pyrene-labeled paromomycin analogs decreased markedly. Moreover, each data point precisely fits the curve obtained from an equation derived assuming 1:1 binding stoichiometry (8). The titration results provide dissociation constants for PBP, PAP, and PCP of 235 6 34, 301 6 13, and 263 6 13 nM, respectively. The studies described above show that PBP, PAP, and PCP show appropriate quenching behavior when treated with the RNA construct. In the next series of experiments, competition assays were set up between the three analogs and paromomycin, an aminoglycoside known to bind to the A-site of the decoding region (7). These competition assays are essential for the success of any high-throughput screen based on the quenching assays. An important issue to address here is whether complete fluorescence recovery occurs when competing aminoglycosides are added. If nonactive site interactions do not occur between the fluorescent moi-
FIG. 1. (A, B, and C) Fluorescence spectra of PBP, PAP, and PCP solutions (10 nM) alone (1) and in the presence of 100 nM (2), 200 nM (3), 300 nM (4), 400 nM (5), 500 nM (6), 600 nM (7), 700 nM (8), 800 nM (9), 900 nM (10), and 1000 nM (11) of RNA.
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cin tested (2 mM). Using PAP (Fig. 4), approximately 90% recovery is observed. On the other hand, the fluorescence intensity of PCP completely recovered to its initial value in the presence of high concentrations of paromomycin, as shown in Fig. 5. Therefore, PCP shows no measurable nonactive site binding due to the pyrene moiety and was thus chosen as the most promising tracer for use in the screening of molecules targeted against the 16S rRNA decoding region. Previously we showed that tobramycin binds to the an HIV–RRE IIB construct of the RRE transcriptional activator region (8). It was of interest to determine if PCT (Scheme 1) would be spectroscopically as wellbehaved in this system as PCP is in the decoding region construct. As shown in Fig. 6A, PCT can be titrated using the RRE construct, providing a dissociation constant of 0.114 6 0.006 mM for the probe. Importantly, when PCT–RRE complexes were titrated with tobramycin, complete recovery of fluorescence was observed (Fig. 6B) and a dissociation constant of 1.56 6 0.62 mM could be calculated for tobramycin. Therefore, PCT shows precisely the kind of fluorescent spectroscopic behavior required of an analog to be used in competitive screening assays. Drug Screening Using the Fluorescence Plate Reader The next step to take in establishing a high throughput fluorescence assay involves adapting the quenching assay described using PCP to a 96-well plate format A solution was prepared containing 500 nM of PCP and 1 mM of the 16S rRNA construct in buffer. Each well of the plate contained 200 mL of above solution and 100 mM of an aminoglycoside aliquot to be tested. The fluorescence emission intensity of PCP, in the well containing 1 mM of the 16S rRNA analog, recovered to FIG. 2. (A, B, and C) Fluorescence spectra of PBP, PAP, and PCP solutions (10 nM) containing 1 mM of RNA and in the absence (1) and in the presence of 10 mM (2), 20 mM (3), 30 mM (4), 40 mM (5), 50 mM (6), 60 mM (7), 70 mM (8), 80 mM (9), 90 mM (10), and 100 mM (11) of paromomycin.
TABLE 1
Dissociation Constants for the Binding of Aminoglycosides to the 16S rRNA Construct i
internal consistency. Furthermore, the dissociation constants measured for paromomycin using these pyrene derivatives are also close to one obtained by a fluorescence anisotropy assay using a different probe (CRP, Scheme 1) (15). While the dissociation constants obtained using PBP and PAP are close to the value obtained using CRP, the fluorescence intensities using the pyrene analogs did not completely recover to their initial values in the presence of high concentrations (2 mM) of paromomycin, suggesting some possible nonspecific binding of the pyrene moiety to RNA in these instances. In the case of PBP (Fig. 3), approximately 90% fluorescence recovery is obtained at the highest concentration of paromomy-
[RNA] 5 1 mM o
i[paromomycin] 5 2 mM/io
Kd (mM)
KD (mM)
0.39 0.31 0.35 —
1 0.9 0.9 —
0.235 6 0.034 0.301 6 0.013 0.263 6 0.013 0.165 6 0.031
2.14 6 0.57 1.29 6 0.21 2.65 6 0.88 1.85 6 0.32
/i
PCP PAP PBP CRPa
Note. io, fluorescence intensity of pyrene-labled paromomycin solution (10 nM) alone. i[RNA] 5 1 mM, fluorescence intensity of pyrenelabled paromomycin solution (10 nM) in the presence of 1 mM of the 16s rRNA analog. i[paromomycin] 5 2 mM, fluorescence intensity of pyrene-labled paromomycin (10 nM) containing 1 mM of the 16s rRNA analog, in the presence of 2 mM of paromomycin. Kd, dissociation constants between the pyrene-labeled paromomycin and the 16S rRNA analog. KD, dissociation constants between paromomycin and the 16S rRNA analog obtained by each tracer. a Previously published in Ref. (8).
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diethylethylglucosamine, and N-heptyl N-hexyl-paraaminoaniline, which should not bind to the decoding region construct, do not compete with PCP as revealed by this assay. DISCUSSION
Since RNA molecules can form intricate three-dimensional structures, it ought to be possible to design small organic molecules to bind to functional domains of RNA. Aminoglycoside antibiotics serve as starting points for design because they have been shown to have a general affinity for RNA molecules (16 –20). With a few notable exceptions (15, 20), the affinity of aminoglycosides for RNA molecules is in the micromole range (16 –19). As more is understood concerning the nature of the specific interactions between aminoglycosides and target RNA species, it should be possible to design higher affinity analogs.
FIG. 3. (A) Relative fluorescence intensity of PBP (10 nM) solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PBP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
varying degrees in the presence of 100 mM of the selected aminoglycosides. Figure 7 show the results of the fluorescence recovery after the addition of the aminoglycosides. In the presence of 100 mM of neomycin, the fluorescence intensity of PCP recovered to 60% of its original intensity. In the presence of paromomycin, kanamycin B, gentamycin, and tobramycin only 30 to 40% fluorescence recovery occurred. By contrast, the fluorescence recovery of PCP caused by the addition of hygromycin B and streptomycin proved to be negligible. These results are consistent with our previous results. The 16S rRNA decoding region analog binds neomycin 10 times tighter than the other aminoglycosides. Furthermore, the construct does not bind either hygromycin B or streptomycin as revealed by fluorescence anisotropy assays (15). In addition, many diverse amine containing molecules, including glucosamine, spermine, dopamine, 4-methoxytryptamine, 1-b-N,N-
FIG. 4. (A) Relative fluorescence intensity of PAP (10 nM), solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PAP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
FLUORESCENCE ASSAYS TO STUDY RNA ANTAGONISTS
189
in the 1 mM range (8). As expected, neither hygromycin nor streptomycin measurably bind (8). In the current work, a fluorescence-quenching assay was developed using the pyrene-labeled paromomycin analogs PBP, PAP, and PCP to monitor the binding of aminoglycosides to the decoding region construct. The fluorescence emission of all three analogs was readily quenched with the decoding region RNA, allowing direct binding constants to be calculated for the three analogs. However, only PCP showed 100% fluorescence recovery upon the addition of high concentrations of paromomycin. In addition, the dissociation constant for added paromomycin in the competition assay using PCP was measured to be 2.14 6 0.57 mM, a value within experimental error of the dissociation constant measured using a fluorescence anisotropy assay and a different fluorescent probe (8). Hence, PCP was chosen as the probe most applicable for use in the screening assay.
FIG. 5. (A) Relative fluorescence intensity of PCP (10 nM), solution as a function of increasing concentrations of the 16S rRNA analog. (B) Relative fluorescence intensity of PCP (10 nM) solution containing 1 mM of the 16S rRNA analog as a function of increasing concentrations of the paromomycin concentration.
There are several naturally occurring RNA constructs of substantial interest with respect to RNA antagonist design. The prokaryotic 16S decoding region and the HIV–RRE transactivator domain are two of these targets. Simple constructs from both RNA regions bind aminoglycosides (6 – 8, 12). In the studies reported here, it was of interest to develop fluorescence-based screens for targets of this type to enable the discovery of new antagonists of biologically interesting RNA targets. The work is largely focused on the decoding region construct. It had previously been shown that the 129-nt decoding region construct under consideration here stoichiometrically binds aminoglycoside antibiotics with the expected affinities. That is, neomycin B binds with a dissociation constant of approximately 0.1 mM, and other aminoglycosides, including tobramycin, gentamycin, and paromomycin, have dissociation constants
FIG. 6. (A) Relative fluorescence intensity of PCT (10 nM) solution as a function of increasing concentrations of the RRE IIB RNA analog. (B) Relative fluorescence intensity of PCT (10 nM) solution containing 1 mM of RRE IIB RNA analog as a function of increasing concentrations of the paromomycin concentration.
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Fig. 4 that PCT would be an ideal candidate for screening studies aimed at discovering novel antagonists of the RRE region of RNA. The experiments reported here show that fluorescent ligands which bind to RNA with fluorescence quenching can be used in the design of rapid and quantitative screening assays for uncovering RNA antagonists. ACKNOWLEDGMENTS These studies were partially funded by U.S. Public Health Service National Institutes of Health Grants EY-03624 and EY-04096. K.H. was funded by a research fellowship from the Japan Society for the Promotion of Science.
REFERENCES FIG. 7. Fluorescence recovery by competitive binding of aminoglycosides. io, fluorescence intensity of PCP (500 nM). iRNA, fluorescence intensity of PCP (500 nM) containing 1 mM of RNA. i, fluorescence intensity of PCP (500 nM) containing 1 mM of RNA in the presence of 100 mM of the aminoglycosides.
PCP was added to the wells in a 96-well plate format along with the decoding region construct. Various aminoglycosides, which were already known to bind to the A-site of the decoding region with varying affinities (8), were then added and the recovery of fluorescence was determined. The results from the screening assay were exactly as predicted from previous studies in which precise determinations of dissociation constants between aminoglycosides and the decoding region construct were made (8). This demonstrates that PCP can be used to access the affinities of analogs directed against the A-site of the decoding region. The screen described here does not, of course, provide an exact dissociation constant for a particular molecule which antagonizes PCP action. Molecules which prove to have activity in this screen can then be quantitatively assayed by standard fluorescent methods (8) to determine precise dissociation constants and stoichiometry of binding. Determination of the stoichiometry of binding is especially important for accessing the specificity of action of an RNA antagonist. The 96-well plate screen is currently being used in our laboratory to uncover novel structural entities capable of competing with aminoglycoside for binding to the decoding region. While PCT was not investigated as thoroughly with respect to the HIV–RRE region as was PCP investigated as a probe for the decoding region, it is clear from
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