Nucleobase Amino Acids and Their Binding Properties to the P22

The specific interaction between proteins and .... Kd is the dissociation constant between the peptide .... tides with the RNA were determined by fluo...
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Bioconjugate Chem. 2004, 15, 694−698

De Novo Design of Peptides with L-r-Nucleobase Amino Acids and Their Binding Properties to the P22 boxB RNA and Its Mutants Hideo Miyanishi, Tsuyoshi Takahashi,* and Hisakazu Mihara Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan. Received November 12, 2003; Revised Manuscript Received April 26, 2004

A method to design novel molecules that specifically recognize a structured RNA would be a promising tool for the development of drugs or probes targeting RNA. In this study, the de novo design of the R-helical peptides having L-R-amino acids with nucleobases (nucleobase amino acids, NBAs) was carried out. Binding affinities of the peptides for a hairpin RNA derived from P22 phage were dependent on the types and positions of the NBA units they have. Some NBA peptides bound to the wild-type RNA or its mutant with high affinity and high specificity compared with the native P22 N peptide. These results indicate that the NBA units on the peptides interact with the RNA bases in a specific manner. It is demonstrated that the de novo design of peptides with the NBA units is an effective way to construct novel RNA-binding molecules.

INTRODUCTION

RNA-protein interactions play important roles in organisms. The specific interaction between proteins and RNA regulates many biological functions, including transcription, RNA splicing, translation and so on (1). In many cases, RNA-binding proteins recognize a specific site of RNA such as hairpin loops, internal loops, and bulges (1). Therefore, construction of the novel molecules that specifically recognize a structured RNA could lead to the development of drug design and specific probes against RNA. However, construction of RNA-binding molecules is difficult compared to that of DNA-binding molecules because of the structural diversity of RNA (2). On the other hand, we utilized artificial amino acids having nucleobases in their side chain (nucleobase amino acids, NBAs) (Figure 1a) for constructing novel functional peptides and proteins (3-5). It has been reported that the NBA units were incorporated into the designed R-helix peptides and the nucleobase interaction on the peptides can regulate the peptide-peptide recognition effectively (3). In the design of RNA-binding molecules, NBAs were studied as building blocks capable of interacting with RNA bases specifically (4, 5). For instance, NBA units were incorporated into the HIV-1 nucleocapsid protein NCp7, and their binding affinities for the HIV-1 Ψ-RNA SL3 region were evaluated (5). An NCp7 protein containing two NBA units at the specific sites showed a higher binding affinity and specificity than the wild-type NCp7. The study demonstrated the applicability of the NBA units as building blocks for constructing RNAbinding molecules. The peptide and protein engineering studies derived from native proteins showed an advantage for designing more specific molecules than the natural counterparts. However, this protein-engineering strategy has the limitation that a lot of proteins with NBAs are difficult to be prepared easily with chemical methods. Furthermore, it is hard to design RNA-binding molecules target* To whom correspondence should be addressed. Fax: +45924-5833. E-mail; [email protected].

Figure 1. (a) Structures of the NBA units. (b) Amino acid sequences of the de novo designed peptides.

ing a variety of RNAs by using natural proteins. On this standpoint, we have attempted to start the de novo design of artificial NBA peptides with simple amino acid sequences in short length. A variety of peptides with various sequences are more easily synthesized than the larger protein. Four kinds of NBA units (adenine, guanine, thymine, and cytosine) could be placed on various positions of peptides capable of forming secondary structures, such as an R-helix and a β-structure. A strategy using de novo design has a high probability of constructing the molecules targeting a variety of structures and sequences of RNA. To approach this goal, in this study, we have performed de novo design of the NBA peptides

10.1021/bc034210n CCC: $27.50 © 2004 American Chemical Society Published on Web 07/02/2004

De Novo Design of Peptides

that have high binding affinities and specificities for a hairpin RNA. The findings in this study lead to a way for constructing novel molecules working against RNAs. MATERIALS AND METHODS

Chemicals and Reagents. All chemicals and solvents were of reagent or HPLC grade. Amino acid derivatives and reagents for peptide synthesis were purchased from Watanabe Chemical (Hiroshima, Japan). MALDI-TOF MS was measured on a Shimadzu MALDI III mass spectrometer by using 3,5-dimethoxy-4-hydroxycinnamic acid as a matrix. HPLC was carried out on a YMC ODS A-302 5C18 column (4.6 mm × 150 mm; YMC, Tokyo, Japan) or a YMC ODS A-323 5C18 column (10 mm × 250 mm) by employing a Hitachi L-7000 HPLC system. Amino acid analyses were performed by using the phenyl isothiocyanate (PTC) method on a Wakopak WS-PTC column (Wako Chemical; Osaka, Japan). Synthetic DNA templates were purchased from Sigma Genosys (Ishikari, Japan). Fluorescein-labeled DNA was purchased from Espec Oligo Service (Tsukuba, Japan). A RiboMAX largescale RNA production system T7 was purchased from Promega (Tokyo, Japan). T4 RNA ligase was purchased from Takara Bio (Osaka, Japan). Peptide Synthesis. Fmoc-protected NBA monomers (Fmoc-GNBA-OH, Fmoc-CNBA(Z)-OH, Fmoc-ANBA(Z)-OH, Fmoc-TNBA-OH) were synthesized according to the reported methods (4b). The peptides were synthesized by the solid-phase method using the Fmoc strategy with O-(7-azabezotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) or 2-(1H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole hydrate (HOBt‚H2O) as the coupling reagents (6). To synthesize the acetylatedpeptide resin, the Fmoc-deprotected peptide resin was reacted with acetic anhydride (10 equiv) in N-methylpyrrolidone (NMP) for 20 min. To remove the resin and the protecting groups, the peptide resin was treated with trifluoroacetic acid (TFA) containing m-cresol, triethylsilane, and thioanisole as scavengers for 1 h at room temperature. For the peptide resin with the Z groups on ANBA and CNBA, the reaction was carried out for 2.5 h. The product was solidified with diethyl ether in an ice bath. All crude peptides were purified with RP-HPLC on a YMC ODS A-323 column using a linear gradient of 8-38% acetonitrile/0.1% TFA (30 min) to give the purified peptides. Peptides were identified by their molecular ion peak (M + H)+ on MALDI-TOF MS: m/z found (calcd) P22N, 2234.0 (2232.6); A9R4, 1324.7 (1324.7); GCG, 1774.8 (1774.0); GTG, 1788.0 (1788.0); GAG, 1797.8 (1798.0); GGG, 1812.9 (1814.0); CGG, 1774.3 (1774.0); TGG, 1789.0 (1788.0); AGG, 1796.7 (1798.0); GGA, 1796.9 (1798.0); AlaGG, 1649.9 (1650.9); GAlaG, 1652.1 (1650.9); GGAla, 1652.7 (1650.9). Preparation of Fluorescein-Labeled P22 boxB RNA and Mutants. The P22 boxB RNA was prepared by transcription of a synthetic DNA template (5′-AATTTAATACGACTCACTATAGGTGCGCTGACAAAGCGCGCC-3′ and 5′-GGCGCGCTTTGTCAGCGCACCTATAGTGAGTCGTATTAAATT-3′) by using the RiboMAX largescale RNA production system T7 (Promega). The template DNA was degraded by RNase-free DNase I, and then the RNA was purified by polyacrylamide gel electrophoresis (PAGE) (15% acrylamide). The fluorescein-labeled DNA oligomer was incorporated into the product RNA using T4 RNA ligase in Tris-HCl buffer (50 mM, pH 7.5) that contained MgCl2 (10 mM), 1,4-dithiothreitol (DTT, 10 mM), adenosine triphosphate (ATP, 1 mM), bovine serum

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albumin (BSA, 0.05%), and dimethyl sulfoxide (DMSO, 10%). The solution was incubated at 15 °C for 16 h. The fluorescein-labeled boxB RNA was purified by ethanol precipitation and PAGE. The mutant RNAs were also prepared using the same method. Concentrations of RNAs were determined by the absorbance at 260 nm. CD Measurements. CD spectra of the peptides were measured on a Jasco J-720WI spectropolarimeter using a quartz cell with 1.0 mm path length in the region of 195-250 nm. Peptides were dissolved in 10 mM TrisHCl buffer (pH 7.5) containing 100 mM KCl and 1 mM MgCl2 or trifluoroethanol (TFE) solution (peptide concentration of 10 µM). Fluorescence Anisotropy Measurements. Fluorescence anisotropy measurements were carried on a Shimadzu RF-5300PC spectrofluorophotometer using a quartz cell with a 1.0 cm path length at 10 °C in N-(2-hydroxylethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES)KOH buffer (10 mM, pH 7.5) containing KCl (100 mM), MgCl2 (1 mM), and EDTA (0.5 mM). Fluorescence anisotropy was calculated by the intensities at 520 nm excited at 480 nm. Determination of Dissociation Constants between Peptides and RNAs. The binding affinities of the peptides for the RNA were determined by fluorescence anisotropy measurements. The fluorescein-labeled RNA (5.0 nM) in the buffer solution was titrated with a peptide solution. After each addition of a peptide, samples were stirred for 30 s and equilibrated for 4.5 min at 10°C, and then fluorescence anisotropy was measured. The dissociation constants of the peptides and RNAs were calculated using eq 1 and Kaleida Graph (Synergy Software), assuming a 1:1 stoichiometry: A) (Ab - Af){[F]0 + [P]0 + Kd - (([F]0 + [P]0 + Kd)2 - 4([F]0[P]0)1/2)} 2[F]0

(1)

[F]0 and [P]0 represent the initial concentrations of the fluorescent RNA and a peptide, respectively. A, Ab, and Af are the anisotropy values of each solution, the bound fluorescent RNA, and the free fluorescent RNA, respectively. Kd is the dissociation constant between the peptide and RNA. RESULTS AND DISCUSSION

Design and Synthesis of the Peptides. To achieve the de novo design of NBA peptides that interact with a hairpin RNA, four kinds of NBA units, alanine, and arginine residues were used. A hairpin RNA was selected as a target molecule because of its important functions in nature such as that in some viral RNAs (HIV-1 TAR, λ phage boxB) and ribosomal RNAs (7). A hairpin RNA studied here is derived from the boxB region of phage P22 RNA that contains a stable GNRA-like (G, guanine; N, any bases; R, guanine and adenine; A, adenine) tetraloop structure (Figure 2a) (8, 9). For the design of the NBA peptides targeting the P22 RNA, NBA units were incorporated into positions 3, 6, and 11 of the peptides (Figure 1b). It was expected that the NBA at position 3 could interact with the stem region of the RNA (Figure 2a). In addition, the NBA units at positions 6 and 11 were expected to interact with the bases of the loop region of the RNA. These expectations were based on the structure of the complex between the boxB RNA and P22 N peptide (8), the N-terminal region of P22 phage N protein (10). In this peptide-RNA complex, the peptide forms an R-helical conformation, the amino acid residue

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Miyanishi et al. Table 2. Dissociation Constants (Kd) of the Peptides with the P22 boxB RNA

Figure 2. (a) Secondary structures of the P22 boxB RNA and its mutants (C11U and 7A15U). The fluorescein group was incorporated into each RNA by using T4 RNA ligase. (b) Amino acid sequence of the P22 N peptide (14-30).

peptide

Kd (µM)

relative affinitya

GCG GTG GAG GGG CGG TGG AGG GGA AlaGG GAlaG GGAla P22N A9R4

1.2 ( 0.1 1.9 ( 0.1 0.86 ( 0.03 0.27 ( 0.02 0.85 ( 0.04 0.79 ( 0.04 0.93 ( 0.03 0.32 ( 0.01 0.53 ( 0.02 3.0 ( 0.3 0.57 ( 0.02 0.88 ( 0.03 120 ( 10

100 63 140 444 141 152 129 375 226 40 211 129 1.00

a The relative affinity represents the ratio of dissociation constants between the A9R4 peptide and other peptides to the P22 boxB RNA.

Table 1. r-Helix Content of the Peptides in TFE Solution peptide

R-helicity (%)

peptide

R-helicity (%)

GCG GTG GAG GGG CGG

58 67 58 71 57

TGG AGG GGA P22N A9R4

50 58 72 62 55

at position 3 is located in the major groove region, and the residues at positions 6 and 11 are located near the bases of the loop region of the boxB RNA. According to the findings, a variety of NBA peptides (13AA) with three NBA units at positions 3, 6, and 11 were designed. Each peptide has six alanine and four arginine residues in addition to the NBA units (Figure 1b). Alanine was selected to generate a high ability to form an R-helical structure (11). Arginine residues were incorporated into the positions expected to interact with phosphate groups electrostatically. In addition to these NBA peptides, the peptides with two guanine NBA units and one alanine residue at these positions were designed. These peptides were expected to clarify the contribution of the NBA unit at each position to the RNA-binding. The A9R4 peptide having three alanine residues at positions 3, 6, and 11 was also designed as a reference molecule for evaluating the NBA function. Peptides were synthesized using the Fmoc solid-phase method (6) and purified by reversed-phase HPLC with high purity. The P22 N peptide was also synthesized as a reference molecule (Figure 2b). All peptides were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and peptide concentrations were determined by amino acid analyses. Circular Dichroism Study. The designed peptides were expected to form an R-helical conformation when the peptide bound to the RNA. Circular dichroism (CD) spectra of the peptides were measured in 10 mM TrisHCl buffer (pH 7.5) containing 100 mM KCl and 1 mM MgCl2 or trifluoroethanol (TFE). In the buffer solution, both the peptides with three NBA units and the P22 N peptide did not form an R-helical structure sufficiently. These results should be due to the repulsions between positively charged residues in the peptides. In contrast, all the peptides with three NBA units showed a typical R-helix CD pattern as well as the P22 N peptide in TFE solution. The R-helicities of the peptides were estimated from the molecular ellipticity at 222 nm (12). As shown in Table 1, the R-helix content of the NBA peptides was estimated as around 50-70%. These results indicate that the NBA peptides have potential to form an R-helical

Figure 3. Relative binding affinities between the peptides and the A9R4 peptide with the P22 boxB RNA. KdA9R4/Kdpeptide represents the ratio of the dissociation constants between the A9R4 peptide and each peptide to the P22 boxB RNA.

structure. The previous study demonstrated that the incorporation of one or two NBA units does not disturb the conformational properties of the peptides and proteins (3, 5). Considering that the R-helix content of the A9R4 peptide was estimated as 55%, in this study, incorporations of three NBA units also do not disturb the abilities of the peptides to form an R-helical structure. Binding Analyses of the NBA Peptides with the P22 boxB RNA. To evaluate the binding affinities of the NBA peptides for the boxB RNA, the fluorescein moiety was incorporated into the RNA using T4 RNA ligase (Figure 2b) (13). Dissociation constants (Kd) of the peptides with the RNA were determined by fluorescent anisotropy measurements. In this method, the binding affinity of the A9R4 peptide for the RNA was estimated as the Kd value of 120 µM. Binding affinity of each peptide was compared with that of A9R4 (Table 2 and Figure 3). At first, the binding affinities of the NBA peptides with GNBA at positions 3 and 11 and the several types of the NBA units at position 6 were compared. The GGG peptide having three GNBA units showed a binding affinity with Kd ) 0.27 µM, a value that indicates affinity ca. 450fold higher than that of the A9R4 peptide. Furthermore, the GGG peptide showed a higher affinity (3.5-fold) than the natural counterpart P22 N peptide, which is known to bind to the P22 boxB RNA. In contrast, the peptides having other types of NBA units at position 6 (A, T, and C bases) bound to the target RNA at a similar or lower level of the P22 N peptide. These results strongly suggested that GNBA6 could work more effectively in the interaction with the boxB RNA than the other types of nucleobases.

De Novo Design of Peptides

Next, the binding affinities of the peptides with the various NBA units at position 3 for the boxB RNA were also compared. In this case, the GGG peptide bound to the target RNA more strongly than the other peptides having adenine, thymine, and cytosine NBA units at position 3. These results indicated that GNBA3 could interact with the RNA base more effectively than the other types of NBA units at its position. GNBA11 was also evaluated to interact with the RNA effectively by a comparison of the binding affinities between the GGG and GGA peptides for the RNA. Therefore, the GGG peptide showed the highest affinity for the P22 boxB RNA in the series of de novo peptides. These results indicated that three guanine bases of the GGG peptide could work effectively for the RNA binding. Generally in RNA, guanine base can interact with not only cytosine base by making the Watson-Crick type base pairing but also guanine by making the Hoogsteen type pairing. Therefore, it seems that three guanines of the GGG peptide are able to interact with a G-C base pair on the stem region of the P22 boxB RNA and guanine and cytosine bases on the loop region. The specific interaction may include not only hydrogen bonding but also hydrophobic interaction. The NMR analysis of the P22 N peptideboxB RNA complex revealed that the amino acid residues of the peptide at positions 3, 6, and 11 are located near the C7-G15, G9, and C11 bases of the boxB RNA, respectively (7). Along with these data, it was estimated that the de novo designed NBA peptides might be located at a position similar to that of the P22 N peptide in the peptide-RNA complex when the NBA peptides could take the R-helical conformation by binding to the RNA. In addition, the other peptides with two guanine bases also showed higher binding affinities than the A9R4. This finding indicates that the two guanine moieties interact with the RNA bases in a manner similar to the case of GGG, and another base (A, T, or C) might interact weakly with RNA by a nonspecific manner. To investigate the contribution of each GNBA unit to the RNA-binding, one of the GNBA units of the GGG peptide was replaced by an alanine residue (Figure 1). As a result, the AlaGG and GGAla peptides showed the binding affinities with Kd ) 0.53 and 0.57 µM, respectively, which is ca. 2-fold lower than the GGG peptide. In contrast, the GAlaG peptide that has the alanine residue at position 6 showed significantly lower binding affinity (Kd ) 3.0 µM) than the other peptides. It was implied that GNBA6 contributed to the RNA-binding to a larger extent than the GNBA units at the other positions. These results do not contradict the facts that the GGG peptide with G at position 6 shows significantly higher affinity for the RNA than the peptides with the A, T, and C bases at the position. Moreover, considering that the binding affinities of the AlaGG and the GGAla peptides were decreased compared with the GGG peptide, GNBA3 and GNBA11 are also important for the RNA-binding. Consequently, since three guanine moieties of the GGG peptide interacted with RNA bases effectively, the GGG peptide showed the higher binding affinity for the P22 boxB RNA than the other peptides including P22 N. Binding Analyses of the NBA Peptides with the Mutant RNAs. By using the NBA units and the de novo designed R-helical peptides, we have successfully demonstrated the construction of the novel RNA-binding molecules. For the evaluation of the specificity of the NBA peptides with the P22 boxB RNA, two types of the mutant RNAs (7A15U and C11U) were prepared (Figure 2a). It was expected that the peptide with the NBA units corresponding to the bases of the mutant RNA showed a

Bioconjugate Chem., Vol. 15, No. 4, 2004 697 Table 3. Dissociation Constants of the Peptides with the C11U Mutant RNA peptide

Kd (µM)

KdP22/KdC11U a

GGG GGA P22N

0.41 ( 0.02 0.18 ( 0.02 0.92 ( 0.03

0.66 1.78 0.96

a K P22/K C11U represents the ratio of the dissociation constants d d of each peptide to P22 boxB RNA and the C11U mutant RNA.

Figure 4. (a) Relative binding affinities of the peptides for the P22 boxB RNA and the C11A mutant RNA. KdP22/KdC11U represents the ratio of the dissociation constants of each peptide to P22 boxB RNA and the C11U mutant RNA. (b) Relative binding affinities of the peptides for the P22 boxB RNA and the 7A15U mutant RNA. KdP22/Kd7A15U represents the ratio of the dissociation constants of each peptide to P22 boxB RNA and the 7A15U mutant RNA.

higher affinity for the mutants than for the wild-type boxB RNA. Binding affinities of the GGA and GGG peptides for the C11U RNA were determined using the same strategy in the case of the boxB RNA (Table 3). Figure 4a shows the relative binding affinities of the peptides for the P22 boxB RNA and the C11U mutant RNA. The GGA peptide bound to the mutant RNA (Kd ) 0.18 µM) more strongly than to the boxB RNA (Kd ) 0.32 µM). In contrast, the GGG peptide showed the binding affinity for the C11U RNA as the Kd value of 0.41 µM; this value is inferior to that for the boxB RNA (Kd ) 0.27 µM). Noticeably, the GGA peptide bound to the C11U mutant RNA more strongly than the GGG peptide did, although the opposite result was obtained in the case of the wild-type RNA. Thus, the GGG and GGA peptides showed a selectivity to the wild-type RNA and the mutant RNA, respectively (Figure 4a). These findings suggested that ANBA11 of the GGA peptide could interact more specifically with the U11 base of the RNA than GNBA11 of GGG did, and its specific interaction enhanced the binding affinity for the mutant RNA. On the other hand, the binding affinity of the wild-type P22N peptide for the C11U RNA (Kd ) 0.92 µM) was almost the same as that for the P22 boxB RNA (Kd ) 0.88 µM). That is, the GGG and GGA peptides could recognize the difference between the cytosine base of the wild-type P22 boxB RNA and the uracil of the C11U mutant more efficiently than the P22 N peptide did. In

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Table 4. Dissociation Constants of the Peptides with the 7A15U Mutant RNA peptide

Kd (µM)

KdP22/Kd7A15U a

GGG AGG TGG CGG P22N

0.36 ( 0.01 0.66 ( 0.04 0.79 ( 0.05 0.94 ( 0.05 0.89 ( 0.03

0.75 1.41 1.00 0.90 0.99

a K P22/K 7A15U represents the ratio of the dissociation constants d d of each peptide to P22 boxB RNA and the 7A15U mutant RNA.

the NMR structural analyses, Arg11 of P22 N peptide interacts with the phosphate group and the sugar at C11 of the wild-type RNA in an electrostatic manner and as a van der Waals contact, respectively. These interactions, however, are not specific for the C11 base of the wildtype RNA. By use of the de novo designed NBA peptide GGG or GGA, cytosine 11 or uracil 11 of the wild-type or the C11U mutant RNA, respectively, can be recognized more specifically by the nucleobase moiety at position 11 of these peptides than the arginine residue of native P22 N. The binding affinities of the peptides for the 7A15U mutant RNA were also evaluated (Table 4 and Figure 4b). In this mutant assay, the AGG peptide showed a selectivity for the 7A15U mutant RNA, and the GGG peptide was selective for the wild-type RNA, while TGG, CGG, and P22N did not show the selective binding for the RNA. The AGG peptide showed a higher binding affinity for the 7A15U RNA (Kd ) 0.66 µM) than for the wild-type boxB RNA (Kd ) 0.93 µM). In contrast, the binding affinity of the GGG peptide for the 7A15U RNA (Kd ) 0.36 µM) was slightly lower than that for the wildtype boxB RNA (Kd ) 0.27 µM). The TGG and CGG peptides showed almost the same affinity as a lower level for both the boxB and the 7A15U RNA. These results indicated that GNBA3 could recognize the C7-G15 base pair of the wild-type RNA more effectively than the A7U15 pair of the 7A15U mutant. In contrast, ANBA3 could recognize the A7-U15 pair more effectively than the C7G15. Incorporation of the NBA units corresponding to the RNA bases into the peptides increases the binding affinities and specificities with the RNA. On the other hand, the dissociation constant of the P22 N peptide for the 7A15U mutant was estimated as Kd ) 0.89 µM, a value that is similar to that for the wild-type RNA (Kd ) 0.88 µM). These results of the native P22 were similar to the case of the C11U mutant. That is, the P22 N peptide could not recognize the differences between the C7-G15 and A7-U15 base pairs nor between the C11 and U11 bases. By use of three NBA peptides (GGG, GGA, and AGG), the mutation of the C7-G15 base pair or the C11U of the P22 boxB RNA can be clarified by the difference of their binding affinities. CONCLUSION

To construct the novel molecules that specifically recognize a hairpin RNA, a series of artificial R-helical peptides with three NBA units were synthesized. These peptides were de novo designed to have simple amino acid

sequences. These synthetic peptides showed sufficient potential for forming an R-helical structure. Binding analyses revealed that the NBA units on the peptides could recognize the RNA bases in a specific manner, and some NBA peptides showed selectivity for recognizing the differences between the wild-type and the mutant RNAs. The combined strategy using the NBA units and peptide scaffolds will be developed to design RNA-binding molecules targeting desired sequences and structures of the RNA. LITERATURE CITED (1) Caprara, M. G., and Nilsen, T. W. (2000) RNA: Versatility in form and function. Nat. Struct. Biol. 7, 831-833. (2) Moore, P. B. (1999) Structural motifs in RNA. Annu. Rev. Biochem. 68, 287-300. (3) (a) Matsumura, S., Takahashi, T., Ueno A., and Mihara, H. (2003) Complementary nucleobase interaction enhances peptide-peptide recognition and self-replicating catalysis. Chem.s Eur. J. 9, 4829-4837. (b) Matsumura, S., Ueno, A., Mihara, H. (2000) Peptides with nucleobase moieties as a stabilizing factor for a two-stranded R-helix. Chem. Commun. 16151616. (4) (a) Takahashi, T., Hamasaki, K., Kumagai, I., Ueno, A., and Mihara, H. (2000) Design of a nucleobase-conjugated peptide that recognizes HIV-1 RRE IIB RNA with high affinity and specificity. Chem. Commun. 349-350. (b) Takahashi, T., Hamasaki, K., Ueno, A., and Mihara, H. (2001) Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA. Bioorg. Med. Chem. 9, 991-1000. (5) Takahashi, T., Ueno, A., and Mihara, H. (2002) Nucleobase amino acids incorporated into HIV-1 nucleocapsid protein increased the binding affinity and specificity to a hairpin RNA. ChemBioChem 3, 543-549. (6) Chan, W. C., and White, P. D. (2000) Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Chan, W. C., and White, P. D., Eds.) pp 41-76, Oxford University Press, Inc., New York. (7) Draper, D. E. (1999) Themes in RNA-protein recognition. J. Mol. Biol 293, 255-270. (8) Cai, Z., Gorin, A., Frederick, R., Ye, X., Hu, W., Majumdar, A., Kettani, A., and Patel, D. J. (1998) Solution structure of P22 transcriptional antitermination N peptide-box B RNA complex. Nat. Struct. Biol 5, 203-212. (9) Legault, P., Li, J., Mogridge, J., Kay, L. E., and Greenblatt, J. (1998) NMR structure of the bacteriophage λ N peptide/ boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif. Cell 93, 289-299. (10) Lazinski, D., Grzadzielska, E., and Das, A. (1989) Sequencespecific recognition of RNA hairpins by bacteriophage antiterminatiors requires a conserved arginine-rich motif. Cell 59, 207-218. (11) Marqusee, S., Robbins, V. H., and Baldwin, R. L. (1989) Unusually stable helix formation in short alanine-based peptides. Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290. (12) Scholtz, J. M., Qian, H., York, E. J., Stewart, J. M., and Baldwin, R. L. (1991) Parameters of helix-coil transition theory for alanine-based peptides of varying chain length in water. Biopolymers 31, 1463-1470. (13) Romaniuk, P. J., and Uhlenbeck, O. C. (1983) Joining of RNA molecules with RNA ligase. Methods Enzymol. 100, 52-59.

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