Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

Alexander Litovchick and Jack W. Szostak
PNAS October 7, 2008 105 (40) 15293-15298; https://doi.org/10.1073/pnas.0805837105

  1. Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved August 12, 2008 (received for review June 17, 2008)

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Abstract

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >1013 different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.

Hepatitis C virus (HCV) is a persistent flavivirus that infects ≈3% of the human population, making it the most common chronic blood-borne infection (1). Among six genotypes of the virus, genotype 1 is the most common in Europe and North America (2). Approximately 75% of HCV-infected individuals develop a largely asymptomatic chronic infection, whereas ≈25% of patients eventually develop liver cirrhosis or hepatocellular carcinoma (1). At present, HCV infection is treated by IFN α/ribavirin therapy until sustained viral response (SVR) is reached. This treatment is effective in ≈50% of patients (1, 3). Resistance to IFN and ribavirin (4), and HCV persistence after SVR in the form of “occult” hepatitis C (3, 5) makes HCV frequently incurable. The virus effectively avoids the host immune response and no vaccine for hepatitis C is currently available (6, 7). Significant effort is being put into the development of specifically targeted antiviral therapies for HCV treatment (STAT-C), aimed at different stages of the viral life cycle, including inhibitors of NS3/4 protease, NS5B replicase, etc., as well as discovery of an anti-HCV vaccine based on E1/E2 fusion proteins (7). Emerging drug resistance has already been observed with anti-HCV compounds in clinical trials (4, 7), highlighting the need for new approaches to HCV therapies.

The 340-nt 5′ UTR is among the most conserved parts of the HCV genome. It contains a highly structured internal ribosomal entry site (IRES) (supporting information (SI) Fig. S1A) that mediates the initiation of translation of the viral polyprotein by a cap- and poly(A)-independent mechanism (8, 9). Translation initiation from the HCV IRES does not require the eIF4F complex: the IRES is recognized directly by the 40S ribosomal subunit and eIF3, recruits eIF2/GTP/Met-tRNA, and the resulting 48S complex assembles at the initiation codon (9, 10). It is noteworthy that the pathway of IFN inhibition of viral replication occurs via an IRES-dependent mechanism (11). Both the IRES structure and the mechanism of HCV translation initiation (e.g., 12, 13) have been the subject of intense research in recent years as a therapeutic target (14–17). As an example, the synthetic steroid mifepristone specifically inhibits in vitro translation initiation from the HCV IRES (18). Unfortunately, mifepristone did not meet the efficacy endpoint for treating HCV infection in a Phase II clinical trial (19).

We have attempted to isolate more effective and specific inhibitors of HCV IRES-initiated translation. Here, we describe the selection of high-affinity peptide binders to the HCV IRES from a cyclic peptide-mRNA fusion (20, 21) library of 10 trillion individual sequences. After 11 rounds of selection, we isolated a bicyclic peptide that binds the HCV IRES tightly and specifically, and selectively inhibits the IRES-initiated translation of a reporter gene in vitro.

Results

In Vitro Selection.

The scheme that we used to select for cyclic peptides that bind specifically to the HCV IRES is outlined in Fig. 1. We started the selection with ≈17 μg of a double stranded DNA library (2.5 × 1014 individual sequences) designed to code for his-tagged peptides containing 10 random residues flanked by cysteines (Fig. S1B). After the transcription of mRNA, photo-cross-linking to the peptide-accepting 3′-puromycin oligonucleotide, and purification by denaturing PAGE, ≈7 μg of the mRNA-oligonucleotide-puromycin library was obtained (2 × 1014 individual sequences). After translation, purification, and peptide cyclization, ≈6.5% of the library was converted into mRNA fusions to cyclic peptides, for an initial library complexity of ≈1.3 × 1013 sequences.

Fig. 1.
Fig. 1.

Overview of the mRNA display selection procedure. mRNA is conjugated to an oligonucleotide with a 3′-puromycin residue; in vitro translation results in peptide synthesis and covalent transfer of the peptide to the puromycin. The mRNA displayed peptide is then cyclized by reaction with dibromo-m-xylene, and the mRNA protected by reverse transcription. Cyclic peptides that bind to the HCV IRES are selected, and the attached cDNA amplified and PCR and T7 transcription.

The selection procedure was divided into three phases of gradually increasing stringency (Table S1; SI Appendix). Rounds 1 and 2 were designed to decrease the complexity of the starting library while retaining all possible IRES binders. During these rounds Torula yeast RNA (TYR) was used as a binding competitor, and all column matrix bound material was eluted nonspecifically with NaOH and 8 M urea. Rounds 3–7 were designed to select more specifically for IRES binders by eluting column bound mRNA-peptide fusions competitively with soluble IRES. Following seven rounds of selection, ≈50% of the input library was eluted from IRES column with soluble IRES RNA in 2 h (Fig. S2). The output cDNA of rounds 6 and 7 was cloned and sequenced, revealing the high complexity of the remaining library. We then further increased the stringency of the selection by using additional selection steps in rounds 8–11, aimed at the selection of peptides with slower dissociation rates, and improved IRES selectivity. Cloning and sequencing of the library after round 11 revealed that the sequence 6B4, encoding the peptide MKCSRGIRCAGVLCGSVGHHHHHHHRL, accounted for >30% of the clones. The same sequence was observed in only 2 of 105 sequences from rounds 6 and 7.

Structure of Peptide 6B4.

Inspection of the peptide 6B4 sequence reveals 3 cysteines, of which 2 originate from the library design, and an additional one from the random region. This suggests the possibility of double cyclization of the peptide in the reaction with dibromoxylene, an additional acceptor of the alkylation reaction being one of the histidine moieties (M. C. Hartman, personal communication; ref. 28). To test this hypothesis, the 6B4 peptide was translated in vitro by using the PURE system (23) and the dibromoxylene cyclization reaction was performed during peptide purification on a Ni-NTA column (24). MALDI TOF analysis revealed a molecular weight of 3,054 for the linear peptide (formylated) and 3,258 for the cyclic peptide (6B4C), corresponding to the addition of two xylene moieties to the peptide (Fig. 2A). When the 6B4 peptide was synthesized by F-moc chemistry, it exhibited a similar double-cyclization product after treatment with dibromoxylene on a Ni-NTA resin (Fig. S3). The synthetic 6B4 was not formylated at the N terminus and the molecular weight of the peptide, which is 3,026 for linear form and 3,230 for the cyclic form, is therefore 28 lower than that of the corresponding in vitro translated peptide (Fig. S3).

Fig. 2.
Fig. 2.

MALDI-TOF analysis of 6B4 peptide. (A) Mass spectra of in vitro translated peptides. Red trace: linear 27-mer 6B4, 3,053.85 observed, 3,054.5 expected. Blue trace, cyclic 6B4C, 3,258.6 observed, 3,258.5 expected. The mass peak at 3,495 (+ve ion mode) is the insulin B chain calibration standard. (B) Possible configurations of bicyclic 6B4C peptide, assuming that one cyclization event involves two cysteines, and the second cyclization involves the remaining cysteine and one histidine. Only the C1-C2, C3-H configuration will be cleaved into two separate cyclic peptides by pepsin digestion. Predicted masses are shown. (C) MALDI-TOF mass spectra of synthetic, cyclized 6B4C peptide (top red trace, observed 3,230.48, expected 3,230.5), pepsin-digested 6B4C (blue trace, note absence of uncleaved peptide at mass peak 3,230.5, and presence of predicted C1-C2 fragment (observed 1,495.4, expected 1,495.7) and C3-H fragment (observed 1,753.5, predicted 1,753.8). The greater intensity of the peak corresponding to the N-terminal C1-C2 fragment may reflect enhanced ionization due to the presence of cationic residues in the N-terminal peptide loop. The lower pink trace is the spectrum of a pepsin self-digest, showing that some of the peaks in the peptide digest originate from pepsin (external mass calibration standard with insulin B chain mass peak, 3,495; data not shown).

Configuration of Cyclic Peptide 6B4C.

Because peptide 6B4 contains three cysteine moieties, multiple double-cyclization variants of the peptide are possible, assuming histidine reactivity (Fig. 2B). Examining the sequence of 6B4 we found a unique pepsin cleavage site, GVL, between the second and third cysteines. Only one bicyclic configuration, denoted C1-C2, C3-H (Fig. 2B) would produce two separate cyclic peptides with molecular weights of 1,495.7 and 1,753.8 after pepsin cleavage at pH ≤ 2; no other configuration would generate two separate products. MALDI-TOF analysis confirmed that the mass peak of 3,230 completely disappeared from the spectrum, whereas new peaks of 1,495.4 and 1,753.5 appeared (Fig. 2C). We conclude that the major configuration of the peptide is C1-C2, C3-H, although other variants may be present at substantially lower quantities in the peptide mixture.

Binding to HCV IRES RNA.

Radiolabeled peptide in both cyclic and linear forms was used to measure binding affinity to HCV IRES RNA and other nonspecific RNAs by equilibrium ultrafiltration (Fig. 3A and Table 1; see also Figs. S4 and S5). The Kd of the linear peptide for HCV IRES RNA was 6.5 nM, whereas the Kd of the cyclic peptide was 0.70 nM. Affinities of the cyclic peptide 6B4C to nonspecific RNA targets such as TYR and a 319-nt-long mRNA (CW mRNA) were 140 and 98 nM, respectively.

Fig. 3.
Fig. 3.

Binding of peptides to HCV IRES RNA. Binding was measured by equilibrium ultrafiltration, and plotted as fraction bound vs. concentration of IRES or competitor peptides. (A) Direct binding of 35S-labeled linear (triangles, interrupted line) and cyclic (circles, solid line) 6B4 peptide to HCV IRES RNA, yielding observed Kd of 6.5 ± 1.8 nM (linear peptide) and 0.70 ± 0.14 nM (cyclic peptide). (B) Competitive binding of linear (triangles, interrupted line) and cyclic (circles, solid line) synthetic 6B48 peptide to IRES, measured as competition with the fluorescent Fl-6B4 peptide. Observed IC50s are 32 nM for cyclic 6B48C and 102 nM for linear 6B48. Kd as calculated from competition with 0.625 nM Fl-6B4 peptide and 15 nM IRES RNA (see Materials and Methods and Fig. S7) are 3.3 ± 0.8 nM (linear peptide) and 0.65 ± 0.12 nM (cyclic peptide). Values are the mean and standard deviation of three to five Kd measurements in each experiment.

View this table:
Table 1.

Structure–activity relationship of 6B4 variants

We then prepared a linear fluorescein-labeled peptide Fl-6B4 for use in competitive binding experiments to allow for the measurement of affinities of unlabeled synthetic peptide variants of the selected peptide. Equilibrium ultrafiltration experiments by using Fl-6B4 and varying concentrations of HCV IRE RNA revealed a Kd of 3.0 nM. The Kd values obtained by competitive binding assays for chemically synthesized 6B4 in linear and cyclic form were 3.3 nM and 0.65 nM (Table 1), respectively, similar to the values obtained by the direct binding assay by using radiolabeled 6B4 and 6B4C. We also measured the affinities of 6B4 and 6B4C for RRE and class I ligase RNAs, which were not used during the selection (Table 1). The linear 6B4 peptide exhibited only moderate specificity (8- and 70-fold tighter IRES binding vs. RRE and class I ligase, respectively), whereas the cyclic 6B4C peptide was highly specific (≈150- and 300-fold, respectively).

Inhibition of IRES-Mediated Translation Initiation In Vitro.

To determine whether the selected peptide can specifically inhibit translation initiated from the HCV IRES, we prepared two constructs with Gaussia Luciferase (GLuc) as a reporter gene. In one construct translation of the GLuc gene was placed under the control of the HCV IRES and, in the other construct, translation was controlled by a generic consensus leader sequence carrying a 5′ m7GpppG cap analog (Fig. 4A). In HeLa cell translation extract the IRES-GLuc and the m7GpppG cap analog leader-GLuc mRNA constructs exhibited similar levels of translation, whereas the uncapped mRNA construct was translated very inefficiently (Fig. S6A). The linear and cyclic peptides 6B4 and 6B4C were found to selectively inhibit IRES-initiated translation of GLuc with IC50s of 95 and 64 nM, respectively, at an mRNA concentration of 50 nM (Fig. 4B, Table 1). Translation of capped leader-GLuc was not inhibited by up to 5 μM peptide (Fig. 4C). Thus, the inhibition of translation by 6B4C in vitro was specific for IRES-initiated translation. As a positive control for both experiments we used 1–2 μM mifepristone, which has previously been shown to inhibit HCV IRES-mediated translation (18). Mifepristone produced ≈50% inhibition of Gaussia Luciferase translation from the IRES-GLuc construct while not affecting the translation of the leader-GLuc constructs.

Fig. 4.
Fig. 4.

Selective inhibition of IRES driven translation by linear and cyclic 6B4 and 6B48 peptides. (A) Schematics of Gaussia Luciferase reporter constructs. (Left) HCV IRES-Gluc construct, (Right) control capped Gluc mRNA. (B and C) Translation of IRES-GLuc (B) and capped leader-GLuc (C) in HeLa S10 extract in the presence of linear 6B4 and cyclic 6B4C, plotted as percent of GLuc luminescence relative to the untreated control. (D and E) Translation of IRES-GLuc (D) and capped leader-GLuc (E) in HeLa S10 extract. in the presence of linear 8-mer (6B48) and the cyclized 8-mer (6B48C) plotted as percent of GLuc luminescence relative to the untreated control.

Minimal Active Structure of 6B4 Peptide.

To identify the minimal region of the selected 6B4 peptide responsible for the HCV IRES binding and translation inhibition properties, we performed experiments with shorter synthetic versions of the 6B4 peptide. Based on the results of pepsin digestion of the 6B4C, we synthesized the N-terminal loop of this peptide, an 8-mer KCSRGIRC (referred to as 6B48), and performed the cyclization reaction in solution (to generate 6B48C). We studied the affinity of the purified linear and cyclic peptides for HCV IRES RNA and their potential as inhibitors of IRES-initiated translation (Table 1). The 8-mer peptide was 3- to 5-fold weaker in affinity for the IRES than the full-length 27-mer 6B4 or 6B4C, with a Kd of 17.5 nM in linear and 3.7 nM in cyclic form (Fig. 3B, Table 1). The specificity of this shorter-cyclic peptide is similar to that of full-length 6B4C. Kd measured by competitive binding for 6B48C to a 319-mer mRNA and ribosomal RNA were found to be 161 and 276 nM, respectively. Affinities to RRE RNA and class I ligase were ≈120 nM and 0.8 μM, respectively (Table 1). Both linear 6B48 and cyclic 6B48C peptides specifically inhibit IRES-initiated translation in HeLa extracts (Fig. 4 D and E). The IC50 of linear 6B48 for IRES-initiated translation inhibition was found to be 125 nM, and for 6B48C it was 76 nM, at an mRNA concentration of 50 nM (Table 1, Fig. 4D). The translation of leader-GLuc and capped leader-GLuc constructs was not inhibited in HeLa extracts by up to 2 μM these peptides (Fig. 4E). The similar IC50 values observed for all peptides in translation inhibition assays reflect the fact that the IRES-mRNA construct used in the translation reactions was present at a concentration well above the Kd for the peptides.

Discussion

The isolation of highly specific peptide aptamers directed to pharmacologically relevant RNA targets has been an ongoing challenge. Although high-affinity cationic linear peptides are easily obtained, they are often not very specific for the particular target RNA sequence, because the electrostatic interaction of the cationic side chains with the anionic RNA backbone typically dominates the binding (26). Recent work on cyclic peptides suggests that rigid structures may yield selective binding not dominated by electrostatics (e.g., 29, 30). We have attempted to address the specificity problem by selection from a very large library of cyclic peptide-mRNA fusions under conditions that stringently select against nonspecific binding. We used cyclized peptides to minimize the entropic cost of peptide binding to the target RNA. To ensure high affinity and specificity of the selected peptide aptamers, the binding selection was carried out in the presence of high concentrations of salt and arginine to reduce nonspecific electrostatic interactions, along with high concentrations of competitor RNA to minimize nonspecific binding. We also used competitive elution of the fusions from the selection column with soluble IRES RNA as a further selection for specific binding. After seven rounds of the selection, we observed a large number of peptide sequences (including 6B4) allowing for high-affinity IRES RNA binding (27), and after four more rounds of selection the highly specific peptide 6B4 accounted for 30% of the surviving sequences.

The selected 6B4 peptide has several noteworthy features. Most striking is the presence of a cysteine residue at a position in the peptide derived from the random region of the original peptide library. This additional cysteine moiety allows for double cyclization after reaction with dibromoxylene: two of the cysteines form one loop, whereas the third cysteine and a histidine (28) form the second. A unique pepsin cleavage site in the 6B4 sequence allowed for unambiguous assignment of the structure of the bicyclic 6B4C: the first loop is between the first two cysteines, and the second loop is between the third cysteine and one of the histidine residues of the his-tag. The factors that drive cyclization into this particular structure are unknown, but may include preorganization of the peptide structure, or greater steric accessibility of the N-terminal region of the peptide when the peptide is immobilized on a Ni-NTA resin via its C-terminal his-tag. A chemically synthesized N-terminal 8-mer peptide bound to IRES RNA almost as well as the full-length 27-mer peptide, suggesting that the N-terminal region contains essentially all of the specificity determinants of the selected peptide. The N-terminal 8-mer peptide contains three basic residues, which are likely to contribute to binding by interaction with specific phosphates in the folded RNA structure. The approximately threefold weaker IRES binding of the 8-mer vs. the original 27-mer may reflect the loss of interactions with the his-tag portion of the peptide. The approximately fivefold tighter IRES binding of the cyclic compared with the linear peptides probably reflects an entropic binding advantage for the conformationally constrained cyclic peptides. The cyclic peptides also exhibited much greater IRES specificity than the corresponding linear peptides, based on a comparison of binding to IRES RNA and to two highly structured RNA molecules that were not used in the selection (RRE RNA and class I ligase ribozyme RNA) (Table 1). These observations reinforce the need for cyclization to obtain highly specific RNA-binding peptides.

The cyclic 6B4C peptide was selected solely on the basis of high affinity and specificity binding to the IRES RNA; it is therefore quite striking that binding does indeed lead to the specific inhibition of IRES-mediated translation initiation. This suggests that the IRES RNA contains a highly structured region that is essential for function, and that acts as an epitope that is particularly suitable for binding to a structured ligand. Peptide binding could inhibit translation initiation by simple steric blockade of interaction with the translational apparatus. Alternatively, ligand binding could prevent an essential conformational transition of the IRES RNA. Future experiments will address the site of peptide binding to IRES and the mechanism of inhibition of IRES-mediated translation initiation.

Our results demonstrate that high-affinity, high-specificity peptide aptamers can be isolated from a sufficiently large starting library by in vitro selection as long as stringent and selective enrichment procedures are used. The relatively small size of 6B48 peptide (Mr of 923 for the linear and 1,025 for the cyclic form), makes it a promising molecule for further pharmacological optimization.

Materials and Methods

HCV IRES RNA.

Cloned HCV IRES of genotype 1b (nucleotides 40–372) was a gift from J. Doudna (Berkeley). The 5′-terminal 40 nucleotides (a stem-loop) were added by PCR to ensure cotranscriptional folding of IRES RNA. A minimum of 1 mM Mg2+ was maintained in all IRES dilution buffers to stabilize the folded state of the RNA. Twenty-three nucleotides were added by PCR on the 3′ end of the construct to ensure synthesis of the fully functional IRES (Fig. S1A). IRES RNA (nucleotides 1–395), including the complete HCV 5′ UTR and 54 nucleotides of the HCV coding region, was prepared by in vitro transcription by using T7 RNA polymerase. The immobilized IRES RNA selection column was generated by transcription of IRES 1–395 with GTP-γ-S followed by covalent attachment to iodoacetyl-activated cross-linked acrylamide resin (Pierce); the resin was then quenched by reaction with mercaptoethanol. The concentration of IRES immobilized on the column was estimated by immobilization of radiolabeled IRES to be 7.5–10 nmol/ml of resin. HIV RRE RNA and the class I ligase RNA are described in the SI Text.

Selection Library Synthesis.

The DNA library included a random 30-nt region flanked by cysteine codons (see Fig. S1B for details) and was synthesized by the Keck facility at Yale University. Transcription, in vitro translation, and mRNA-peptide fusion formation were done essentially as described in ref. 21 with minor modifications as follows. For round 1, 2 nmol of cross-linked mRNA was translated in vitro in 4 ml of wheat germ (WG) extract (Promega), instead of 10 ml of RRL, for 1 h at 30°C. Cyclization was performed on an oligo(dT) cellulose (NEB) column equilibrated with cyclization buffer [660 mM KCl, 20 mM Tris, pH 8.0, 0.2 mM TCEP, and 3 mM R,R′-dibromo-m-xylene (Aldrich) in 30% acetonitrile/70% water mixture] and incubated for 1 h with gentle shaking (22). The cyclized fusions were eluted, concentrated, and then purified on a Ni-NTA column (Qiagen) under denaturing conditions (21). The purified fusions were ethanol precipitated and reverse transcribed (Fig. S1B). In subsequent rounds, translation and fusion formation were performed in 1–2 ml of WG extract and all purification procedures were done in proportionally smaller volumes.

Selection of IRES RNA Binders.

Cyclic peptide-mRNA fusions were applied to an IRES selection column and incubated for 15–20 min in selection buffer S (20 mM Hepes, pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.05% Tween 20, 10 units/ml RNasin), supplemented with different concentrations of NaCl, arginine, and Torula Yeast RNA (TYR) (see below and Table S1). The column was washed with 10–20 column volumes of the same buffer and then eluted. The eluted material was PCR-amplified and used to initiate the next round of selection (summarized in Table S1).

In rounds 1 and 2 buffer S was supplemented with 0.75 M NaCl, 10 mM arginine, and 20 μM TYR. After washing, mRNA-peptide fusions captured on the column were eluted by either 10 mM NaOH (round 1) or 8 M urea (round 2). For rounds 3–7, buffer S was supplemented with 0.5 M NaCl, 15 mM arginine, and 80 μM TYR. Preelution was performed in the same buffer with 100 μM TYR for 2 h and this eluate was discarded. Specific elution was performed with 10 μM freshly transcribed soluble IRES in buffer S + 200 mM NaCl for 2–4 h at room temperature. In rounds 8–11, the preelution was performed in buffer S containing 0.5 M NaCl supplemented with up to 30 μg/μl of additional competitor RNA, such as Escherichia coli 16S and 23S RNA (Roche), phenol-extracted rabbit ribosomes, obtained by gel filtration of RRL on a Sepharose-6B column (Aldrich), a 319-nt-long mRNA derived from an unrelated selection, and a Tetrahymena intron RNA in vitro transcribed by T7 RNA polymerase. This eluted material was discarded. The first specific elution was done with 10–12 μM soluble IRES in buffer S + 200 mM NaCl for 1–2 h; this eluate was also discarded. The second specific elution was carried out by using 10 μM IRES in buffer S + 200 mM NaCl for 12–16 h at +4°C and only this eluted sample was used for the initiation of the next round of selection. PCR products obtained after rounds 6, 7, and 11 were cloned into the TOPO-TA vector (Invitrogen) and sequenced. The selection stopped after 11 rounds.

In Vitro Translation and Cyclization of Peptide 6B4.

In vitro translation of the 6B4 peptide was performed in a reconstituted E. coli translation mixture (PURE system; 23) in the presence of 35S methionine, with cyclization on a Ni-NTA column, and analyzed by MALDI TOF as described (24; see SI Text for details). Oxidized insulin chain B (Mr 3,495) was used as a mass standard.

Synthetic 6B4 Peptide.

The 27-residue peptide MKCSRGIRCAGVLCGSVGHHHHHHHRL (6B4), the 8-residue variant of 6B4 referred to as 6B48, KCSRGIRC, and the 27-mer 6B4 labeled at its N terminus with the 6-isomer of fluorescein isothiocyanate (Fl-6B4) were synthesized by using F-moc chemistry and purified by GenScript Corp. Full-length 6B4 was cyclized with dibromo-m-xylene on a Ni-NTA column as described above, producing a bicyclic derivative of the peptide, 6B4C. The mass spectra of the peptides were determined by MALDI-TOF MS (see SI Text for details).

Pepsin Digestion of 6B4C.

Linear 6B4 and bicyclic 6B4C peptides were digested to completion with 0.1% pepsin for 30 min at 30°C in 0.1% TFA at pH ≤ 2. The reaction was desalted and concentrated by using C18 Zip Tips (Millipore) and was analyzed by MALDI-TOF.

Solution Binding Assays.

The 35S-labeled peptide 6B4 was synthesized by in vitro translation, cyclized on a Ni-NTA column when necessary, desalted on a Sephadex G-10 spin column, and purified on a PepClean C-18 spin-column (Pierce). For each data point, 200 μl of 1 nM linear 6B4 or 0.5 nM cyclic 6B4C peptide in buffer (20 mM Tris·HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2, 0.05% Triton X-100) was incubated for 1 h with freshly transcribed and purified HCV IRES RNA. RNA concentration was measured by UV absorption (Cary UV spectrometer). Equilibrium ultrafiltration measurements of dissociation constants were performed as described in ref. 25.

The fluorescein-labeled peptide Fl-6B4 was used as a probe for solution binding and competition experiments (see SI Text for details). A sample of 200 μl of 2 nM Fl-6B4 in buffer (20 mM Hepes, pH 7.4, 300 mM NaCl, 5 mM MgCl2, 2 mM CaCl2, 0.025% Triton X-100, and 0.5% DMSO), was incubated for 1 h with a series of increasing concentrations of HCV IRES RNA. Equilibrium ultrafiltration was performed by using YM-30 spin filters (Millipore) and fluorescence spectra of top and bottom chambers were collected on a Varian Cary Eclipse spectrofluorimeter (see Fig. S3 for examples). The Kd of Fl-6B4 was calculated as described in ref. 25.

For competitive binding experiments, 200 μl of 0.4–0.6 nM Fl-6B4 and 15–18 nM IRES (determined to give ≈70% binding), was preequilibrated for 1 h in binding buffer, then increasing concentrations of competing peptides were added and incubated for an additional 1 h. Samples were then treated as described above (see SI Text for details).

IRES-Reporter Gene Constructs.

The HCV IRES (1–371) sequence, including 30 nucleotides of the core protein coding region, was added in frame to a Gaussia luciferase (GLuc, NEB) reporter gene. Control reporter gene constructs were prepared by PCR by using the same GLuc sequences downstream of a 32-mer leader sequence (Fig. 4A). The constructs were PCR amplified and used for the transcription of mRNA by T7 RNA polymerase. Capped constructs were prepared by transcription in the presence of 10 mM cap analog m7GpppG (NEB).

HeLa S10 Extract Preparation and in Vitro Translation in HeLa S10.

HeLa S10 translation extract was prepared as described in ref. 14 from a 6-ml HeLa S3 cell pellet obtained from the National Cell Culture Center. HeLa cell extract translation reactions were carried out as described in ref. 14. For the measurement of luciferase activity, 50-μl reactions containing 10–50 nM reporter construct mRNAs were incubated for 1 h at 30°C. Different concentrations of peptides were premixed with measured amounts of mRNA before addition to in vitro translation extracts. For the visualization of GLuc translation, the Renilla luciferase assay kit (Promega) was used, because coelenterazine is the substrate for both Renilla and Gaussia luciferases. Samples of 10–15 μl of translation reactions were transferred into black 96-well plates (Corning) and mixed with an equal volume of 1× lysis buffer. The coelenterazine solution in the assay buffer was added and the light output was measured on a TopCount NXT luminometer plate reader (Perkin–Elmer).

Acknowledgments

We thank J. Doudna for the generous gift of HCV IRES construct; C-W. Lin for the CW mRNA, help with the PURE system, peptide synthesis, and purification; Z. Mujawar for the pNL4–3 plasmid; D. Shechner for the class I ligase RNA; J. Doudna, R. Green, C-W. Lin, D. Treco, Q. Dufton, M. C. Hartman, F. Seebeck, Y. Guillen, B. Seelig, R. Bruckner, A. Bell, and A. Luptak for helpful discussions. This work was supported by the HHMI. J.W.S. is an Investigator and A.L. was an Associate of the Howard Hughes Medical Institute.

Footnotes

  • *To whom correspondence should be addressed. E-mail: szostak{at}molbio.mgh.harvard.edu

  • Author contributions: A.L. and J.W.S. designed research; A.L. performed research; A.L. and J.W.S. analyzed data; and A.L. and J.W.S. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0805837105/DCSupplemental.

  • Freely available online through the PNAS open access option.

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Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display
Alexander Litovchick, Jack W. Szostak
Proceedings of the National Academy of Sciences Oct 2008, 105 (40) 15293-15298; DOI: 10.1073/pnas.0805837105
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