NOVEMBER/DECEMBER 2002 Volume 13, Number 6 © Copyright 2002 by the American Chemical Society
COMMUNICATIONS A New Class of RNA-Binding Oligomers: Peptoid Amide and Ester Analogues Venkitasamy Kesavan,† Natarajan Tamilarasu,† Hong Cao, and Tariq M. Rana* Chemical Biology Program, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605-2324. Received June 28, 2002
Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′-end of all HIV mRNAs. Here we report the design, synthesis and in vitro activities of oligopeptoid amide and ester analogues which bind TAR RNA with high affinities. These results show that we have identified a new class of unnatural oligomers for RNA targeting.
RNA-protein interactions are involved in many cellular functions such as transcription, RNA splicing, and translation. Small peptides with unnatural backbones that can bind with high affinity to a specific sequence or structure of nucleic acids and interfere with proteinnucleic acid interactions would provide useful tools in molecular biology and medicine. Successful approaches used thus far include duplex-forming (antisense) (1) and triplex-forming (anti-gene) oligonucleotides (2-4), peptide nucleic acids (PNA) (5), and pyrrole-imidazole polyamide oligomers (6, 7). Each class of compounds employs a readout system based on simple rules for recognizing the primary or secondary structure of a linear nucleic acid sequence. Another approach employs carbohydrate-based ligands, calicheamicin oligosaccharides, which interfere with the sequence-specific binding of transcription factors to DNA and inhibit transcription in vivo (8, 9). While * To whom correspondence should be addressed. Phone: (508) 856-6216. Fax: (508) 856-8015. E-mail: tariq.rana@ umassmed.edu. † These authors contributed equally to this work.
antisense oligonucleotides and PNA employ the familiar Watson-Crick base-pairing rules, two others, the triplexforming oligonucleotides and the pyrrole-imidazole polyamides, take advantage of straightforward rules to read the major and minor grooves, respectively, of the double helix itself. In addition to its primary structure, RNA has the ability to fold into complex tertiary structures consisting of such local motifs as loops, bulges, pseudoknots, and turns (10, 11). It is not surprising that, when they occur in RNAs that interact with proteins, these local structures are found to play important roles in protein-RNA interactions (12). This diversity of local and tertiary structure, however, makes it impossible to design synthetic agents with general, simple-to-use recognition rules analogous to those for the formation of double- and triple-helical nucleic acids. Since RNA-RNA and proteinRNA interactions can be important in viral and microbial disease progression, it would be advantageous to have a general method for rapidly identifying synthetic compounds for targeting specific RNA structures. A particu-
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Figure 1. (A) Secondary structure of TAR RNA used in this study. TAR RNA spans the minimal sequences that are required for Tat responsiveness in vivo (27) and for in vitro binding of Tat-derived peptides (28). Chemical syntheses were performed on ABI synthesizer Model 392 using standard protocols. TAR RNA was chemically synthesized on a fluorescein-containing CPG 500 support. Synthesis of RNA, deprotection, characterization, and purification were performed as described earlier (20, 22). (B) Schematic comparison of peptides and peptoids.
lar protein-binding RNA structure can be considered as a molecular receptor not only for the protein with which it interacts but also for synthetic compounds, which may prove to be antagonists of the protein-RNA interaction. The mechanism of transactivation of human immunodeficiency virus type 1 (HIV-1) gene expression requires the interaction of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′-end of all nascent HIV-1 transcripts (13). Inhibition of Tat-TAR interactions is a potential approach for anti-HIV therapeutics. Here we report that two oligopeptoids bind TAR RNA with high affinities in vitro. We have recently begun to examine TAR RNA recognition by unnatural biopolymers containing urea and carbamate backbone structures (14, 15). Small Tatderived oligocarbamate and oligourea bind TAR RNA specifically with high affinities in vitro (14, 15). TAR RNA-binding oligocarbamate and oligourea are also resistant to protease degradation in vitro (14, 15). While devising oligomers alternative to the natural polymers, the following points should be taken into consideration: (i) Monomers should be straightforward to synthesize in large amounts, (ii) the monomers should have wide variety of functional groups presented as the side chains of the oligomeric backbone, and (iii) the linkage should be resistant to hydrolytic enzymes. To effect the above attributes, we planned to synthesize peptoid analogue oligomers (16-18). The schematic comparison of peptides and peptoids (Figure 1B) shows the similarities in the spacing of the side chains and carbonyl groups and the differences in the chirality of two monomers. Similarities between peptides and peptoids should not obscure the important differences in stereochemical and conformational characteristics of the two oligomers. In addition, peptoids are stable to hydrolysis by proteases (19). We envisioned that modification of oligopeptoids with ester or amide moieties would change the physical and chemical properties of the oligomers that could result in enhanced bioavailability and in vivo stability. We synthesized oligopeptoids containing basic arginine-rich region of Tat through solid-phase synthesis (Scheme 1). Rink-amide MBHA resin was treated with piperidine and then coupled with bromoacetic acid in the presence of diisopropylcarbodiimide. Bromide was displaced with ester or amide of the corresponding amino
Scheme 1. The Synthesis of Oligopeptoid Ester and Amide Analogsa
a (a) 20% piperidine/DMF; (b) bromoacetic acid/diisopropylcarbodiimide, DMF; (c) methyl ester or amide of amino acid, diisopropylethylamine, DMF.
acid in the presence of diisopropylethylamine. These cycles were repeated to synthesize Tat-derived oligopeptoids containing amide or ester groups (Figure 2). For the oligopeptoid ester synthesis, rink amide resin (0.1 mmol) was swelled in dimethylformamide (2 mL) for 15 min. After filtration, 1 M bromoacetic acid in DMF (2 mL) and diisopropylcarbodiimide (400 µL) were added. The resin was filtered after 2 h and washed with DMF (5 × 3 mL). Bromide displacement was effected in the presence of side chain protected amino ester (1 mmol) and triethylamine (1 mmol) in DMF for 12 h. The above procedure was repeated with bromoacetic acid followed by respective amino ester to obtain Tat-derived peptoid ester oligomer. After the completion of synthesis, the resin was cleaved with a mixture of TFA (95%), triisopropylsilane (2.5%), and water (2.5%) and purified by HPLC. The peptoid ester oligomer was characterized by MALDI mass spectrometry (calculated mass for M + H ) 2352.5; observed ) 2353.8). Similar procedures were used to synthesize the amide analogues of the Tat-derived peptoids. For dye labeling, MBHA resin containing oligopeptoid amide (14.4 µmol) was treated with 5-(and-6)-carboxytetramethylrhodamine (150 µmol) and diisopropylcarbodiimide (150 µmol) for 3 h. The resin was washed repeatedly with DMF and dichloromethane. Rhodaminelabeled Tat-peptoid was cleaved from the resin by treating with trifluoroacetic acid, water, and phenol (95:2.5: 2.5). The product was purified by reverse phase HPLC and characterized by MALDI mass spectrometry (calculated mass ) 2378.2; observed ) 2376.26).
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Figure 2. (A) Amino acid sequence of a Tat derived peptide (47-57) that contains RNA-binding region of HIV-1 Tat protein. Structures of the carboxytetramethylrhodamine-labeled oligopeptoid amide (B) and the tyrosine containing oligopeptoid ester (C). Side chains corresponding to the Tat peptide are shown in (A).
Figure 3. TAR RNA-binding affinities of oligopeptoid amide by FRET (A) and by fluorescence quenching (B). The fluorescence resonance energy transfer (FRET) from fluorescein to rhodamine was measured on PTI (Photon Technology International) fluorescence spectrophotometer. The fluorescein-labeled TAR RNA (Fl-TAR) was excited at 490 nm. The emission spectrum was recorded from 500 to 650 nm for each scan. The slits were set to 4 nm for both excitation and emission lights. All experiments were carried out at room temperature. The initial concentration of Fl-TAR (3′-end fluorescein labeled TAR RNA) was 5.2 nM for all experiments. Each addition of rhodamine-labeled oligopeptoid amide to the Fl-TAR solutions was followed by a 2-min equilibration before the fluorescence spectrum was recorded. For fluorescence quenching experiments, unlabeled oligopeptoid ester was added, the spectra were recorded as described above for the oliogpeptoid amide, and KD values were determined by fitting data to quadratic eq 3.
To determine the TAR RNA binding activities of Tatderived oligopeptoids, we employed fluorescence spectroscopy techniques. Modified TAR RNA was synthesized on an Applied Biosystems Model 392 DNA/RNA synthesizer using 2-cyanoethylphosphoramidite chemistry (Figure 1A). All the monomers of (2-cyanoethyl)phosphoramidites were obtained from Glen Research (Sterling, VA). TAR RNA was chemically synthesized on a fluores-
cein-containing CPG 500 support. RNA (1 µmol) containing fluorescein was deprotected by treatment with NH3 saturated methanol (2 mL) at 25 °C for 17 h. Product was filtered and dried in SpeedVac. To deprotect 2′-OH silyl groups, the pellet was dissolved in 50% triethylamine trihydrofluoride in dimethyl sulfoxide (0.5 mL) and left at room temperature for 16 h. Deprotected RNA was precipitated by the addition of 2 mL of isopropyl alcohol.
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After deprotection, RNA was purified and characterized as described previously (20-22). This fluorescein-labeled TAR RNA was used to perform fluorescence resonance energy transfer (FRET) and fluorescence quenching experiments. We carried out FRET experiments to determine the dissociation constant of TAR RNA and peptoid-amide complex. FRET, in which a fluorescent donor molecule transfers energy via a nonradiative dipole-dipole interaction to an acceptor molecule (which is usually also a fluorescent molecule) is a standard spectroscopic technique for measuring distances in the 10-70 Å range (23, 24). The donor’s lifetime and intensity are reduced upon energy transfer, and the acceptor fluorescence is increased, or sensitized. Quantification of the efficiency of energy transfer allows determination of the distance between the two fluorophores. We used a well-characterized donor-acceptor dye-pair, fluorescein-rhodamine, for FRET experiments. Rhodamine was incorporated at the terminal amino groups of the oligopeptoid amide during solid-phase synthesis. The fluorescence resonance energy transfer (FRET) from fluorescein to Rhodamine was measured on PTI (Photon Technology International) fluorescence spectrophotometer. The fluorescein-labeled TAR RNA (Fl-TAR) was excited at 490 nm. The emission spectrum was recorded from 500 to 650 nm for each scan. The slits were set 4 nm for both excitation and emission lights. All experiments were carried out at room temperature. Samples were placed in a plastic microcuvette with a starting volume of 300 µL. The initial concentration of Fl-Tar was around 5.2 nM for all experiments. Each addition of rhodamine-labeled Tat peptoid amide to the Fl-TAR solutions was followed by a 2-min equilibration before the fluorescence spectrum was recorded. The fluorescence intensities were calibrated for the background of buffer (TKT buffer: 50 mM Tris-HCl, pH ) 7.4, 20 mM KCl, and 0.02% (w/w) Tween20). The energy transfer efficiency (E) from the donor to the acceptor was calculated from the decrease of the donor fluorescence intensity at 516 nm according to eq 1:
E ) (1 - IDA/ID)
(1)
where IDA and ID are the donor fluorescence intensities in the presence and absence of the acceptor, respectively. The distance between the donor and acceptor (R) was calculated according to eq 2:
R ) R0(E-1 - 1)1/6
(2)
where R0 ) 47.6 Å for fluorescein-rhodamine pair in our system (25). The dissociation constant (KD) of oligopeptoid amide bound to TAR RNA was obtained by fitting data to quadratic eq 3:
F ) Fmin -{(Fmax - Fmin)[(e0 + x + KD)((e0 + x + KD)2 - 4e0x)1/2]}/2e0 (3) where F is the relative fluorescence intensity of the donor (Fl-TAR). Fmin is the fluorescence intensity at the start of the titration. Fmax is the fluorescence intensity at saturating concentration of oligopeptoid amide, x. e0 is the initial concentration of Fl-TAR, 5.2 nM. Results of these experiments are shown in Figure 3A indicating that a high affinity complex between the Tat-derived peptoid
amide analogue and TAR RNA was formed (KD ≈ 155 nM). The energy transfer efficiency was 47.6%, and a distance of 48.4 Å was calculated between two dyes. We were unable to incorporate rhodamine at the amino terminal position during the synthesis of Tat-derived oligopeptoid ester analogue due to the formation of a terminal diketopiperazine under basic conditions (diisopropylamine) during rhodamine dye labeling reactions. Therefore, we used fluorescence quenching of fluoresceinTAR RNA by the addition of oligopeptoid ester and determined the binding affinities of Tat-oligopeptoid ester to TAR RNA. As shown in Figure 3B, the oligopeptoid ester was able to bind TAR RNA with high affinity (KD ≈ 68 nM). It is interesting that the ester analogue of the Tat-derived oligopeptoid displays a higher affinity to TAR as compared to the amide analogue which contains more functional groups for nucleic acid backbone interactions. Future structural studies may elucidate the differences in RNA recognition by these two oligopeptoids. Wender et al. recently demonstrated the use of peptoids as molecular transporters (26). Peptoid transporters proved to be substantially better than Tat (49-57) peptide. In addition to better uptake performance, these peptoids offer several advantages over Tat peptides including ease of synthesis of analogues and protease stability. These features along with their significant water solubility (>100 mg/mL) indicate that the peptoids could serve as drug candidates, effective transporters for the molecular delivery of drugs and other agents into cells. Our findings reported here show that two analogues of oligopeptoids (ester and amide) derived from Tat sequence binds TAR RNA with high affinities. RNA recognition by an oligopeptoid provides a new tool for the design of drugs and cellular probes which could modulate RNA-protein interactions in vivo. Studies to analyze cellular uptake and in vivo activities of these oligopeptoids are in progress. ACKNOWLEDGMENT
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