Modified peptide selection in vitro by introduction of a protein–RNA interaction

Shinya Y. Sawata1 and Kazunari Taira1,2,3

1Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562 and 2Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-8656, Japan

3 To whom correspondence should be addressed. e-mail: taira{at}chembio.t.u-tokyo.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
The ribosome display system is a very effective and powerful tool for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. The system depends on the stability of ribosome–mRNA complexes that have been formed as a result of the removal of a stop codon. To assess the general applicability of the system, we examined the stability of ribosome–mRNA complexes in the presence and absence of a stop codon, as well as in the presence and the absence of an additional interaction between the translated peptide and its mRNA within the ribosome–mRNA complex. The additional interaction that we exploited was the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (Cv). The MSp dimer and Cv were placed, respectively, at the N-terminal end of a nascent protein, translated in vitro, and at the 5' end of the protein’s mRNA, and consequently further stabilize the ribosome–mRNA complex. To our surprise, we were able to select proteins even in the presence of a stop codon. Moreover, as we had anticipated, the interaction between the MSp dimer and Cv enhanced the stability of the ribosome–mRNA complex, suggesting that this kind of interaction might be useful in the design of an efficient ribosome display selection strategy. Indeed, the yield of the mRNAs of interest after selection was increased upon the introduction of the interaction between the MSp dimer and Cv.

Keywords: in vitro selection/MS2 coat protein/ribosome display/RNA–protein interaction/stop codon


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Tools for the selection of a protein (or peptide) of interest from a pool of various proteins (or peptides) have recently attracted considerable attention. It is now easy to prepare a pool of high-quality mRNAs whose nucleotide sequences have been artificially randomized or derived by cloning from an organism or cultured cells. Such pools of mRNA are readily converted into pools of proteins (or peptides) by translation in vitro, and the subsequent selection and isolation of the protein or peptide of interest, using a resin with an appropriate immobilized ligand or antibody, are relatively straightforward. However, determination of the sequence of a protein that has been recovered as a result of its specific function by in vitro selection is extremely difficult, unless phenotype and genotype are linked, because the amount of the product translated in vitro is generally very small and amplification is not possible, at least at this time.

One of the most widely used techniques for linking genotype and phenotype is the phage display system (Smith, 1985Go). This system depends on exposure of the protein of interest as a fusion protein with an endogenous capsid protein on the surface of bacteriophage particles. The viral particles retain the gene for the protein of interest within the viral genome so that genotype and phenotype are linked in a single bacteriophage particle. This system involves multiple steps, and success depends on the efficiency of infection of Escherichia coli and the budding activity of bacteriophages. Moreover, proteins that are toxic to cells or that play key roles in cells might not be selectable in such a system, even though many useful proteins have been selected by this method.

To complement selection systems in vivo, selection systems that depend on cell-free translation in vitro have recently been devised (Mattheakis et al., 1994Go; Gersuk et al., 1997Go; Hanes and Plückthun, 1997Go; He and Taussig, 1997Go; Nemoto et al., 1997Go; Roberts and Szostak, 1997Go; Hanes et al., 1998Go, 1999Go, 2000aGo,b; Tawfik and Griffiths, 1998Go; Doi and Yanagawa, 1999Go; He et al., 1999Go; Makeyev et al., 1999Go; Schaffitzel et al., 1999Go, 2001Go; Bieberich et al., 2000Go; Cho et al., 2000Go; FitzGerald, 2000Go; Liu et al., 2000Go; Coia et al., 2001Go; Irving et al., 2001Go; Hammond et al., 2001Go; Jermutus et al., 2001Go, 2002Go; Keefe and Szostak, 2001Go; Lamla and Erdmann, 2001Go; Tabuchi et al., 2001Go; Wilson et al., 2001Go; Zhou et al., 2001Go; Amstutz et al., 2002Go; Fujita et al., 2002Go; Takahashi et al., 2002Go). Cell-free systems require no time-consuming steps such as cell culture, and they do not depend on biological factors, such as efficiencies of infection, which are sometimes responsible for non-reproducible results. Since some of the features of in vitro selection systems, including high reproducibility, are suitable for automated screening, such as high-throughput screening (HTS), in vitro selection systems should effectively complement widely used in vivo systems in future analysis.

The ribosome display system is a representative of systems for in vitro selection of proteins via cell-free translation. The ribosome display system is conceptually very simple and linkage of phenotype with genotype is accomplished simply by removal of a stop codon; the ribosome forms a relatively stable complex with the translated protein and the mRNA that encodes the protein because the rate of release of the protein from the complex is reduced in the absence of the stop codon (Hanes and Plückthun, 1997Go; He and Taussig, 1997Go; Hanes et al., 1998Go, 1999Go, 2000Goa,b; He et al., 1999Go; Makeyev et al., 1999Go; Schaffitzel et al., 1999Go, 2001Go; Coia et al., 2001Go; Irving et al., 2001Go; Jermutus et al., 2001Go, 2002Go; Lamla and Erdmann, 2001Go; Amstutz et al., 2002Go; Takahashi et al., 2002Go).

Most successful experiments using ribosome display have involved the in vitro selection of single-chain variable fragments of antibodies (scFv) (Hanes and Plückthun, 1997Go; He and Taussig, 1997Go; Hanes et al., 1998Go, 1999Go, 2000Goa,b; He et al., 1999Go; Makeyev et al., 1999Go; Schaffitzel et al., 1999Go, 2001Go; Coia et al., 2001Go; Irving et al., 2001Go; Jermutus et al., 2001Go, 2002Go). To examine the stability of the mRNA–ribosome–protein complex in the ribosome display system, and also to examine whether it might be possible to select peptides other than scFv, we chose histidine-tagged peptide that included six-tandem histidine residues as the peptide of interest; this was the selection-positive peptide. We also chose a biochemically meaningless peptide derived from a plasmid vector as the selection-negative peptide. We then prepared two kinds of mRNA, one including a stop codon (i.e. ‘stop codon plus’) and one lacking a stop codon (i.e. ‘stop codon minus’) in its open-reading frame (ORF) by in vitro transcription of the respective histidine-tag-coding and non-coding genes.

Since the success of the ribosome display system depends heavily on the stability of the mRNA–ribosome–protein complex, we postulated that additional interactions between the mRNA and its translated product might improve the efficiency of selection in the ribosome display system. To examine this possibility, we chose the MS2 coat protein (MSp) and the RNA motif to which it binds, namely, C-variant (Cv), which originated from E.coli bacteriophage MS2 (Golmohammadi et al., 1993Go; LeCuyer et al., 1995Go, 1996Go; Ni et al., 1995Go; Peabody, 1997Go; Valegård et al., 1997Go; Convery et al., 1998Go; Johansson et al., 1998Go; Rowsell et al., 1998Go; van den Worm et al., 1998Go). The MSp and Cv were placed at the N-terminal end of the nascent protein and at the 5' end of the mRNA, respectively. We postulated that the binding of MSp to Cv might increase the stability of the mRNA–ribosome–nascent protein complex.

Recently, we developed other systems for protein selection in vitro. The first one was the Ribosome-Inactivation Display System (RIDS), which was dependent on the deceleration of the translation by means of ricin (Zhou et al., 2001Go). The second one was dependent on the stable interaction between a tat peptide and an RNA aptamer of the tat peptide (Fujita et al., 2002Go). The ability of ricin was limited to the inactivation of the eukaryotic ribosomes, and also, the complex of the tat peptide and the RNA aptamer was not formed effectively in the E.coli extract of the prokaryotic translation system. Therefore, these systems were not suitable for the investigation of the prokaryotic proteins. The system reported here was expected to compensate for this disadvantage because the MSp originated from bacteriophage MS2.

We report here that (i) in contrast to our expectations, selection of the peptide of interest was possible even in the presence of a stop codon and (ii) the additional MSp–Cv interaction did indeed improve the yield of selection-positive mRNA.


    Materials and methods
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Preparation of a mutant gene for MSp (mMSp)

A lysate of Saccharomyces cerevisiae L40 ura MS2 strain (Invitrogen, Carlsbad, CA, USA) included the gene for MSp and we used the lysate as the source of template DNA for PCR. We subcloned the amplified gene for MSp and used the resultant genome as template for mutagenesis by PCR, which allowed us to introduce the desired mutations into MSp, namely, valine (V) 75 to glutamic acid 75 (E), alanine (A) 81 to glycine (G) 81, and arginine (R) 82 to tryptophan (W) 82. These mutations yielded MSp that was not incorporated into capsids by self-assembly, but did not eliminate its RNA-binding ability (LeCuyer et al., 1995Go).

Preparation of the Cv motif

The Cv motif is an RNA motif that binds to mutant MSp (mMSp) mentioned above (LeCuyer et al., 1995Go; Valegård et al., 1997Go; Convery et al., 1998Go; Johansson et al., 1998Go; Rowsell et al., 1998Go). We prepared this motif as double-stranded DNA from two chemically synthesized DNA oligomers. The sense sequence was 5'-TCG AGA CAT GAG GAT CAC CCA TGT G-3' and the antisense sequence was 5'-TCG ACA CAT GGG TGA TCC TCA TGT C-3'. To clone the Cv motif into the vector, we included an EcoRI site and a SalI site upstream and downstream of the artificial double-stranded DNA, respectively.

Construction of plasmids

The original plasmid was prepared by cloning of the gene for dihydrofolate reductase (dhfr) from E.coli into the multicloning site of pET30-a(+) (Novagen, Inc., Madison, WI, USA). The plasmid was designated pXH(+)D, where H(+) refers to a histidine tag. We expected the dhfr to act only as a spacer peptide. Next, we generated a plasmid, designated pXH(–)D, which did not have a sequence encoding a histidine tag. Plasmid pXH(–)D was prepared by removal of the region between the NdeI and NspV sites and replacement of this region by a synthesized double-stranded DNA linker that did not encode a histidine tag. The sequence of the linker DNA oligomers were 5'-TAT GTC TGC TAA ATT-3' (sense) and 5'-CGA ATT TAG CAG ACA-3' (antisense). Plasmids pXH(+)D and pXH(–)D were the parent plasmid of all plasmids that we constructed and utilized in the present study. All plasmids with a histidine tag-coding sequence were derived from pXH(+)D and all plasmids without such a sequence were derived from pXH(–)D. Thus, H(+) and H(–) in the names of plasmids refer to the presence and absence of a histidine tag-coding sequence, respectively. All Cv-containing plasmids were derived from pXH(+)D or pXH(–)D. As mentioned above, the double-stranded DNA fragment containing the Cv motif had EcoRI and SalI sites at its termini. Plasmids, pXH(+)D and pXH(–)D had EcoRI and SalI sites between their T7 promoter and Shine–Dalgarno (SD) sequences. Therefore, we were able to insert the Cv motif between the sites of initiation of transcription and translation by cloning the DNA fragment that contained the Cv motif between the EcoRI and SalI sites of these vectors. The terms Cv and X in names of plasmids refer to the presence and absence of a Cv motif, respectively. We cloned the gene for mMSp at the PstI site in the plasmids.

Preparation of template DNA for transcription in vitro

We employed the two pairs of DNA oligomers as primers for PCR in order to prepare template DNA for transcription in vitro. The correlations of template DNA with plasmids are shown in Table I. The upstream primer in the first pair was designated fP-1 and the downstream primer was designated rP-1. The sequence of fP-1 was 5'-GCG TAG AGG ATC GAG ATC GA-3' and that of rP-1 was 5'-CCG GAT ATA GTT CCT CCT TTC-3'. When the plasmids discussed above were employed as templates for PCR with this pair of primers, the PCR products contained a T7 promoter in the upstream region and a stop codon downstream of the longest ORF. The second pair of primers was fP-1 and rP-2, whose sequence was 5'-GTT ATT GCT CAG CGG TGG CA-3'. After PCR with this second pair of upstream and downstream primers and these plasmids mentioned above as templates, the PCR products each contained a T7 promoter in the upstream region but no stop codon within the longest ORF. The conditions for PCR were as follows in all cases: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 74°C for 1 min. The cycle was repeated 30 times and the final volume of each reaction mixture, which contained Ex TaqTM (Takara Shuzo Co., Ltd, Japan), was 200 µl. The PCR products were purified with a QIAquick® PCR purification kit (Qiagen, GmbH, Hilden, Germany), and concentrations of purified products of PCR were estimated from the absorption at 260 nm of each solution. Each template DNA was named according to rules for naming plasmids (Table I). Inclusion of ‘d’ in the name of a construct means that it was the product of PCR that was prepared as template DNA. Inclusion of ‘D(–)’ in the name of a construct means that there was no stop codon within the longest ORF (i.e. gene for the dhfr) in the PCR product.


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Table I. The correlation between plasmid, DNA and RNA prepared for this investigation
 
Preparation of mRNA for in vitro translation

The correlations of mRNA transcribed in vitro with template DNA are shown in Table I. Transcription in vitro was performed with a RiboMAXTM Large Scale RNA Production System (Promega Corporation, Madison, WI, USA) according to the general protocol supplied with the kit. The concentration of template DNA was 1 µg per 50 µl of reaction mixture. After in vitro transcription, deoxyribonuclease I (Takara Shuzo Co., Ltd) was added to the reaction mixture, which then was incubated at 37°C for 1 h. The transcribed RNA was purified with an RNeasy® Mini kit (Qiagen) and the concentration of purified RNA was estimated from the absorption at 260 nm. Each mRNA was named according to the rules for naming plasmids (Table I). An ‘r’ in the name of a construct indicates an mRNA prepared for in vitro transcription. A ‘D(–)’ in the name of a construct refers to the absence of a stop codon within the longest ORF. For internal labeling with radioactive phosphate, in vitro transcription was performed as above, with the addition of 0.74 MBq of [{alpha}-32P]cytidine 5'-triphos phate ([{alpha}-32P]CTP) (NEN® Life Science Products, Inc., Boston, MA, USA) to the reaction mixture.

Translation in vitro

We used an E.coli S30 extract for linear templates (Promega Corporation) as the in vitro translation system. We predicted the sizes of the mRNA that encoded the histidine tag and the mRNA that did not from each encoded amino acid sequence, and we estimated the difference between them. We made the same calculations for the mRNA with and without the Cv motif. Our estimates indicated that we could ignore these differences because they were relatively small. We used 2 µg of input mRNA when the mRNA was not radiolabeled. In the case of the comparative analysis of mRNAs whose lengths were significantly different from one another, for example in the assay for which results are given in Figure 7, more complex considerations were necessary to eliminate artifacts owing to stoichiometric factors. Details of these conditions are discussed below. Transcripts were translated according to the general protocol from Promega. First, we prepared a mixture of 5 µl of complete amino acids mixture, which included 1 mM of each amino acid (Promega), 0.5 µl of a solution of 10 mg/ml rifampicin (Sigma), 20 µl of the S30 premix without added amino acids that was included in the translation kit, 15 µl of the S30 extract included in the translation kit, and appropriate amounts of the solution of mRNA and RNase-free water. This mixture was kept on ice until the start of the translation reaction, which was initiated by floating the sample tube in a water bath that had been adjusted to 37°C. Translation was allowed to proceed at 37°C for 10 min according to protocols in the literature (Hanes and Plückthun, 1997Go; Schaffitzel et al., 1999Go; Lamla and Erdmann, 2001Go).



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Fig. 7. (A) Estimation of the contribution of ribosome–mRNA interaction for the in vitro selection. The vertical scale shows the radioactivity of internally radiolabeled mRNAs remaining on the Ni-NTA agarose beads just prior to the elution step of the in vitro selection. (B) Correlation between inclusion in the system of the mMS2p–Cv interaction and the yield of in vitro selection. The vertical scale shows the yield, which was defined as the ratio of radioactivity associated with pelleted Ni-NTA agarose before the elution step of selection to the radioactivity of the input radiolabeled mRNA. The numbers below the horizontal axis are the operation number of two individual experiments.

 
Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of [35S]methionine-labeled proteins

Radiolabeling of translated products was achieved by two changes in the protocol for in vitro translation described above. We used 1 mM amino acids mixture minus methionine (Promega) instead of the complete amino acids mixture and added [35S]methionine (NEN® Life Science Products, Inc.) to the reaction mixture prior to in vitro translation. The radiolabeling reaction was stopped by placing the sample tube on ice and adding 50 µl of sample buffer for SDS–PAGE [0.1 mM Tris–Cl, pH 6.8, 4% (w/v) SDS, 12% (v/v) ß-mercaptoethanol, 20% (v/v) glycerol, plus bromophenol blue], and then the sample was heated at 100°C for 5 min. The radiolabeled sample was loaded onto an SDS–polyacrylamide gel (15% polyacrylamide) and electrophoresis was continued until the bromophenol blue had reached the bottom of the gel. After electrophoresis, the gel was exposed to an imaging plate (type BAS-III; Fuji Photo Film, Co., Ltd, Tokyo, Japan) for 12 h in darkness. The resultant image was analyzed by the STORM 830 system with ImageQuaNTTM software (Molecular Dynamics, Inc., Sunnyvale, CA, USA).

Selection in vitro

We examined various buffer solutions in an effort to optimize selection and finally settled on three buffers. The binding buffer was prepared by mixing solutions as follows: 420 µl of distilled water, 25 µl of 1 M K2HPO4 (pH 9.4), 50 µl of 0.5 M KH2PO4 (pH 5.6), 300 µl of 1 M NaCl, 5 µl of 10% (v/v) polyoxyethylene(20) sorbitan monolaurate (Wako Pure Chemical Industries, Ltd, Osaka, Japan), 100 µl of 20% (w/v) Block AceTM solution (Dainippon Pharmaceutical Co., Ltd, Osaka, Japan), and 100 µl of 0.5 M Mg(CH3COO)2. Block Ace is a blocking reagent used for the western blotting assay, similar to sterilized milk, to decrease non-specific binding of ribosome complexes to target molecules as well as to the control surface. The advantages of such reagents as sterilized milk were described previously (Hanes et al., 1998Go). The selection buffer was a mixture of 430 µl of binding buffer and 20 µl of a suspension of nickel nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen). The elution buffer was prepared by mixing solutions as follows: 270 µl of distilled water, 25 µl of 1 M K2HPO4 (pH 9.4), 50 µl of 0.5 M KH2PO4 (pH 5.6), 300 µl of 1 M NaCl, 5 µl of 10% (v/v) polyoxyethylene(20) sorbitan monolaurate, 100 µl of 20% (w/v) Block AceTM solution and 250 µl of 1 M imidazole. After 50 µl of the solution of in vitro translated transcript, which was prepared according to the same protocol as the in vitro translation mentioned above, had been mixed with 450 µl of selection buffer, the mixture was incubated at room temperature for 10 min with gentle and repeated inversion of the sample tube. We called this step the binding step since the goal was the binding of the histidine tag of the translated product to Ni-NTA that was immobilized on agarose beads. The binding step was followed by washing, as follows. The sample was centrifuged at 2000 g for 1 s and then the supernatant was carefully removed. Then, 200 µl of binding buffer were added to the Ni-NTA agarose beads, which had been pelleted by centrifugation, with gentle mixing. The washing step was repeated three times. After the third removal of the supernatant, 200 µl of elution buffer were added to the pelleted Ni-NTA agarose beads. The mixture was incubated at room temperature for 10 min with repeated gentle inversion of the sample tube, as described above. Then, the Ni-NTA agarose beads were pelleted by centrifugation at 2000 g for 1 s and the supernatant, containing the eluted mRNA of interest, was carefully removed to a new microtube. The mRNA in this solution was purified with an RNeasy® Mini kit. The purified mRNA was reverse-transcribed by M-MLV reverse transcriptase (Promega) to yield complementary single-stranded DNA. We designed a single-stranded DNA oligomer as the primer for reverse transcription (RT), which we designated rP-3; its sequence was 5'-CTT GTC GTC GTC GTC GGT-3'. A mixture of 10 µl of the solution of purified mRNA and 4 µl of 1 mM rP-3 was incubated at 70°C for 5 min and then placed on ice for 1 min. The sample containing mRNA and the annealed RT primer was mixed with 1 µl of PCR nucleotide mix (Promega) which was a premixed solution of 10 mM of each dNTP in water, and 4 µl of optimized buffer for RT (Promega). Then, 1 µl of the solution of M-MLV reverse transcriptase was added to the sample, which was kept on ice until the reaction was started by floating the tube in a water bath adjusted to 42°C. The RT reaction was allowed to proceed for 2 h and was followed by PCR. The volume of the reverse-transcribed sample used as the template for PCR was 2 µl. We used two DNA oligomers as primers for PCR, namely fP-2 (5'-GGA TAA CAA TTC CCC TCG AGA-3') and rP-3 (see above). The number of cycles for amplification ranged from 15 to 19. When the template originated from pCvH(+)mM2D and pXH(+)D, the length of the product of RT–PCR was 223 and 198 bp, respectively. When the template originated from pCvH(–) mM2D and pXH(–)D, the length of the product of RT–PCR was 160 and 135 bp, respectively. We identified the origin of each product of RT–PCR from its mobility during agarose gel electrophoresis. To compare the histidine tag-coding gene with the histidine tag-non-coding gene, we used a 2% (w/v) SeaKem® GTG agarose gel. For comparison of products of RT–PCR that originated from pCvH(+)D (223 bp) and from pXH(+)D (198 bp), we used a 3% (w/v) SeaKem® GTG agarose gel. The products of RT–PCR on agarose gels were stained with SYBR® Green I (Molecular Probes, Inc., Eugene, OR, USA). The patterns of bands after electrophoresis were analyzed using Fluor Imager 595 and ImageQuaNTTM software (Molecular Dynamics, Inc.). We designed the forward and reverse primers for amplification of all reverse-transcribed products, and we adjusted the number of cycles from 15 to 17. Therefore, we considered that the ratios of RT–PCR products reflected the relative levels of mRNAs in the pool of recovered mRNA. To confirm the validity of our calculations, we estimated the ratio of the products of RT–PCR that originated from a pool of mRNA that was composed of a mixture of equal amounts of histidine tag-coding mRNA and histidine tag-non-coding mRNA. We found that, as anticipated, the ratio of the amount of product derived from histidine tag-coding mRNA to that derived from histidine tag-non-coding mRNA was 1:1.

Comparative analysis of the efficiency of the selection system

To estimate the efficiency of in vitro selection, we used internally radiolabeled mRNA and carried out the selection protocol as described above but without the elution step. Then, we measured the radioactivity associated with the pelleted Ni-NTA agarose beads instead of analyzing the mRNA by RT–PCR and agarose gel electrophoresis. We confirmed initially that the background radioactivity of the pelleted Ni-NTA agarose beads was close to zero. Thus, the radioactivity that remained associated with the Ni-NTA agarose beads after the in vitro selection reflected the amount of mRNA recovered during selection. Thus, this assay allowed us to estimate the efficiency of the in vitro selection system. For a comparative analysis of mRNAs of significantly different lengths as, for example, in the assay for which results are given in Figure 7A, we attempted to eliminate artifacts due to stoichiometric factors as follows. We chose one mRNA as the standard for the normalization. We then estimated the cytidine content per molecule of each mRNA and calculated the theoretical ratios of cytidine content relative to that of the mRNA chosen as the standard. Then, we adjusted the relative radioactivity (c.p.m.) of each input mRNA by reference to the theoretical relative cytidine contents estimated as indicated above. Such normalization allowed us to perform in vitro translation and in vitro selection under conditions whereby the numbers of molecules of input mRNAs were (theoretically) identical to one another, even when the respective sequences and lengths of the mRNAs subjected to comparisons were significantly different.


    Results and discussion
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 Abstract
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Correlation between the presence of a stop codon and the success of in vitro selection

Since the success of the ribosome display system is heavily dependent on the stability of the mRNA–ribosome–protein complex, and since it has been assumed that such stability is achieved by the removal of the stop codon, we focused initially on the correlation between the presence of a stop codon in the ORF and the success of in vitro selection. We prepared two kinds of plasmid, one with [pXH(+)D] and one without [pXH(–)D] a histidine tag, and subsequently obtained four kinds of mRNA, two with and two without a stop codon (Figure 1A; see also Materials and methods or Table I). The ‘selection-positive’ peptide of interest was the histidine-tagged peptide that included six histidine residues in tandem and the control peptide that lacked the histidine tag was the ‘selection-negative’ peptide.



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Fig. 1. (A) The constructs prepared as templates for in vitro transcription. Blue squares represent a histidine-coding gene and red squares represent a stop codon. We designated these template DNAs: dXH(+)D(+), (I); dXH(–)D(+), (II); dXH(+)D(–), (III); dXH(–)D(–), (IV). Both dXH(+)D(+) and dXH(–)D(+) included a stop codon, while, dXH(+)D(–) and dXH(–)D(–) did not. SD, Shine–Dalgarno sequence. (B) Confirmation of in vitro translation. Lane 1, the translated product of rXH(+)D(+); lane 2, the translated product of rXH(–)D(+); lane 3, the translated product of rXH(+)D(–); lane 4, the translated product of rXH(–)D(–). (C) Semi-quantitative analysis for bands detected in (B) using ImageQuaNTTM software.

 
Each cDNA, furnished with a T7 promoter sequence, was amplified by PCR and the products of PCR were transcribed to yield two kinds of mRNA (with and without the stop codon in the ORF). The expected lengths were 915 [Figure 1A (I)], 852 [Figure 1A (II)], 845 [Figure 1A (III)] and 782 bp [Figure 1A (IV)], respectively. Thus, we obtained a total of four kinds of mRNA: rXH(+)D(+) [Figure 1A (I)]; rXH(–)D(+) [Figure 1A (II)]; rXH(+)D(–) [Figure 1A (III)]; rXH(–)D(–) [Figure 1A (IV)] (Table I). The four kinds of transcribed mRNA were subsequently used for in vitro translation of their encoded proteins (Figure 1B). The predicted molecular masses were 26.5 kDa (lanes 1 and 3 in Figure 1B) and 24.2 kDa (lanes 2 and 4 in Figure 1B), respectively. As shown in Figure 1B, the levels of proteins in lanes 1 and 2 (mRNAs with a stop codon) were apparently greater than those in lanes 3 and 4 (mRNAs without a stop codon).

The result was quantitated using a STORM 830 system and image analysis software (ImageQuaNTTM) as described in Materials and methods (Figure 1C). The differences in levels can be explained by the differences in the rates of turnover of ribosomes during in vitro translation, with a significantly higher turnover when there was a stop codon in the ORF (lanes 1 and 2). The release of the ribosome from the complex, which allows the ribosome to participate in a subsequent cycle, is induced by release factors only in the presence of the stop codon.

We used the four mRNAs for in vitro selection of our target (histidine tag-containing) protein. After the collection of selection-positive mRNAs from the 1:1 mixture of selection-positive and selection-negative mRNAs using Ni-NTA agarose beads, we estimated the efficiency of such in vitro selection by RT–PCR, as shown in Figure 2A. Lanes 4 and 8 show the products of RT–PCR that originated from the 1:1 mixture of the pools of mRNA prior to in vitro selection. These two lanes show unambiguously that the two pools were composed of equal amounts of histidine tag-coding mRNA [+; Figure 1A (I) and (II)] and histidine tag-lacking mRNA [–; Figure 1A (III) and (IV)]. Lanes 1 and 5 show the results for the selection-positive controls [Figure 1A (I) and (III)], and lanes 2 and 6 show the results for the selection-negative controls [Figure 1A (II) and (IV)], which were the products of RT–PCR that originated from individual pools of mRNA composed of each unique sequence (i.e. not mixed mRNA pools).



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Fig. 2. The patterns of products of RT–PCR after agarose gel electrophoresis are shown. (A) Lanes 1–3 and lanes 5–7 show products of RT–PCR amplified from each mRNA pool after in vitro selection. Lane 1, the initial mRNA pool contained only rXH(+)D(+); lane 5, the initial mRNA pool contained only rXH(+)D(–); lane 2, the initial mRNA pool contained only rXH(–)D(+); lane 6, the initial mRNA pool contained only rXH(–)D(–); lane 3, the initial mRNA pool was a 1:1 mixture of rXH(+)D(+) and rXH(–)D(+); lane 7, the initial mRNA pool was a 1:1 mixture of rXH(+)D(–) and rXH(–)D(–); lane 4, the product of RT–PCR derived from a 1:1 mixture of rXH(+)D(+) and rXH(–)D(+) prior to in vitro selection; lane 8, the product of RT–PCR derived from a 1:1 mixture of rXH(+)D(–) and rXH(–)D(–) prior to in vitro selection. (B) The results of in vitro selection in the case where the ratio of the selection positive mRNA to the selection negative mRNA in the initial pool was asymmetric. Lane 1, the initial mRNA pool was a 0.2:1 mixture of rXH(+)D(+) and rXH(–)D(+); lane 3, the initial mRNA pool was a 0.2:1 mixture of rXH(+)D(–) and rXH(–)D(–); lane 2, the product of RT–PCR derived from a 0.2:1 mixture of rXH(+)D(+) and rXH(–)D(+) prior to in vitro selection; lane 4, the product of RT–PCR derived from a 0.2:1 mixture of rXH(+)D(–) and rXH(–)D(–) prior to in vitro selection.

 
Lane 7 shows the results of selection from the mixed pool of mRNAs (shown in lane 8), under conditions similar to those used for the ribosome display method (no stop codon in the ORF). Lane 7 shows that the dominantly selected mRNA from the 1:1 mixture of mRNA pools was the histidine tag-coding mRNA [+; Figure 1A (III)] and it demonstrates the appropriate selection of the target protein in the absence of a stop codon (in the ribosome display system).

The result in lane 3 was, however, unexpected. Lane 3 represents selection using the mixed pool of mRNAs, as shown in lane 4, with the one difference from lane 7 being that, in lane 3, these mRNAs had a stop codon. Lane 3 clearly showed that in vitro selection worked even when there was a stop codon in the ORF and suggested that the mRNA–ribosome–protein complex was relatively stable in the presence of a stop codon, at least under the present conditions.

The initial pool originated from 0.2:1 mixture of selection-positive and selection-negative mRNAs was also investigated. As shown in Figure 2B, it supported our interpretation as described above. To confirm this suggestion and to eliminate potential artifacts, we performed similar selections in the presence of 1 mM EDTA instead of 50 mM Mg(CH3COO)2 since EDTA is known to destabilize the mRNA–ribosome–protein complex. As expected, when there was a stop codon in the ORF, the yield was quite low when the binding buffer contained 1 mM EDTA (see below and Figure 7A).

Therefore, it appeared that successful selection of the target protein in the presence of a stop codon depended on the stability of the mRNA–ribosome–protein complex, and that the linkage between each mRNA and its nascent polypeptide might involve polysomes rather than monosomes, at least under our conditions.

The addition of mutant MSp (mMSp)–Cv interaction for the ribosome display

In an attempt to improve the selection efficiency of our system still further, we introduced an additional interaction between the mRNA and its translated product, using MSp and the Cv RNA motif to which it binds with high affinity (LeCuyer et al., 1995Go; Valegård et al., 1997Go; Convery et al., 1998Go; Johansson et al., 1998Go; Rowsell et al., 1998Go). We placed MSp and Cv at the N-terminal end of the nascent protein and at the 5' end of its mRNA, respectively. We expected that the binding of MSp to Cv would further increase the stability of the mRNA–ribosome–protein complex, as depicted in Figure 3. We postulated that such circularization might also stabilize the mRNA within mRNA–ribosome–protein complexes against degradation by endogenous ribonucleases.



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Fig. 3. A schematic hypothetical representation of the ribosome display containing an additional interaction (mMSp–Cv). This system works under the polysome situation.

 
Structural analysis of the MSp–Cv complex by X-ray diffraction showed that a symmetric dimer of MSp recognized a single RNA molecule with the Cv binding motif (Valegård et al., 1997Go; Convery et al., 1998Go; Rowsell et al., 1998Go), and that the N-terminus of one monomer and the C-terminus of another monomer were located close to one another in the symmetric dimer (Golmohammadi et al., 1993Go). Therefore, we fused two mMSp (mutant MSp: see Materials and methods)-coding genes with a flexible peptide linker and placed these tandemly fused genes for mMSp at the N-terminus of the target protein and the Cv motif at the 5' end of the mRNA. The concept of a tandem dimer of MSp-coding genes itself has been already reported (Peabody, 1997Go).

From a kinetic analysis, the rate of dissociation (koff) of the complex between the mMSp dimer and the Cv motif was 0.0038 min–1 at 0°C (Johansson et al., 1998Go). From these data, we estimated the half-life of the mMSp–Cv complex to be 3 h and postulated that this half-life was sufficient for the in vitro selection.

We prepared plasmids with [pCvH(+)mM2D] and without [pCvH(–)mM2D] a histidine tag, which subsequently yielded four kinds of mRNA, namely, two mRNAs with and two mRNAs without a stop codon, in order to assess the strategy described above (see Materials and methods). All of the mRNAs had a Cv motif at the 5' end and all encoded two copies of mMSp, connected in tandem, at the N-terminus of each ORF. The selection-positive target peptide was the histidine-tagged peptide that included six histidine residues in tandem and the control peptide, derived from the plasmid vector, was the selection-negative peptide. Each sequence furnished with a T7 promoter was amplified by PCR. The expected lengths were 1786 [Figure 4A (I)], 1723 [Figure 4A (II)], 1716 [Figure 4A (III)] and 1653 bp [Figure 4A (IV)], respectively. The resultant products of PCR encoded two kinds of mRNA, namely, mRNAs with or without a stop codon in the ORF, yielding a total of four kinds of mRNA: rCvH(+)mM2D(+) [Figure 4A (I)]; rCvH(–)mM2D(+) [Figure 4A (II)]; rCvH(+)mM2D(–) [Figure 4A (III)]; rCvH(–)mM2D(–) [Figure 4A (IV)] (Table I).



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Fig. 4. (A) The constructs prepared as templates for in vitro transcription to estimate the effect of mMSp–Cv interaction for the ribosome display. Green, blue, purple and red squares represent a Cv motif sequence, histidine-coding gene, mMSp-coding gene and stop codon, respectively. We designated the template DNAs: dCvH(+)mM2D(+), (I); dCvH(–)mM2D(+), (II); dCvH(+)mM2D(–), (III); dCvH(–)mM2D(–), (IV). Both dCvH(+)mM2D(+) and dCvH(–)mM2D(+) had a stop codon, while both dCvH(+)mM2D(–) and dCvH(–)mM2D(–) did not. (B) Confirmation of in vitro translation. Lane 1, the translated product of rCvH(+)mM2D(+); lane 2, the translated product of rCvH(–)mM2D(+); lane 3, the translated product of rCvH(+)mM2D(–); lane 4, the translated product of rCvH(–)mM2D(–).

 
The four kinds of transcribed mRNA were then translated in vitro to yield the respective proteins (Figure 4B). The predicted molecular masses were 55.7 (lanes 1 and 3 in Figure 4B) and 53.5 kDa (lanes 2 and 4 in Figure 4B), respectively. In Figure 4B, the levels of proteins in lanes 1 and 2 (with a stop codon) were detectable, but the corresponding levels in lanes 3 and 4 (without a stop codon) were under the limit of detection. The difference can be explained by the difference in the rate of turnover of ribosomes during in vitro translation, with a significantly higher rate when there was a stop codon in the ORF (lanes 1 and 2).

The four mRNAs were used for in vitro selection of the target (histidine tag-containing) protein. After the collection of the selection-positive mRNA from the 1:1 mixture of the selection-positive and selection-negative mRNAs with Ni-NTA agarose beads, we estimated the performance of the selection process by RT–PCR, as shown in Figure 5A. Lanes 4 and 8 show the products of RT–PCR that originated from the 1:1 mixture of the mRNA pools prior to in vitro selection. These lanes show unambiguously that the mRNA pools were composed of equal amounts of histidine tag-coding mRNA [+; Figure 4A (I) and (II)] and histidine tag-non-coding mRNA [–; Figure 4A (III) and (IV)]. Lanes 1 and 5 show the results for selection-positive controls [Figure 4A (I) and (III)], and lanes 2 and 6 show the results for selection-negative controls [Figure 4A (II) and (IV)], namely products for RT–PCR that originated from individual mRNA pools composed of unique sequences (i.e. not mixed mRNA pools).



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Fig. 5. (A) The results of in vitro selection, performed to estimate the efficiency of our novel system. The patterns of products of RT–PCR agarose gel electrophoresis are shown. Lanes 1 –3 and lanes 5–7 show products of RT–PCR amplified from each mRNA pool after in vitro selection. Lane 1, the initial mRNA pool included only rCvH(+)mM2D(+); lane 5, the initial mRNA pool included only rCvH(+)mM2D(–); lane 2, the initial mRNA pool included only rCvH(–)mM2D(+); lane 6, the initial mRNA pool included only rCvH(–)mM2D(–); lane 3, the initial mRNA pool was a 1:1 mixture of rCvH(+)mM2D(+) and rCvH(–)mM2D(+); lane 7, the initial mRNA pool was a 1:1 mixture of rCvH(+)mM2D(–) and rCvH(–)mM2D(–); lane 4, the product of RT–PCR amplified from a 1:1 mixture of rCvH(+)mM2D(+) and rCvH(–)mM2D(+) before in vitro selection; lane 8, the product of RT–PCR amplified from a 1:1 mixture of rCvH(+)mM2D(–) and rCvH(–)mM2D(–) prior to in vitro selection. (B) The results of in vitro selection in the case where the ratio of the selection positive mRNA to the selection negative mRNA in the initial pool was asymmetric. Lanes 1 and 3 show products of RT–PCR amplified from each mRNA pool after in vitro selection. Lane 1, the initial mRNA pool was a 0.2:1 mixture of rCvH(+)mM2D(+) and rCvH(–)mM2D(+); lane 3, the initial mRNA pool was a 0.2:1 mixture of rCvH(+)mM2D(–) and rCvH(–)mM2D(–); lane 2, the product of RT–PCR derived from a 0.2:1 mixture of rXH(+)D(+) and rXH(–)D(+) prior to in vitro selection; lane 4, the product of RT–PCR derived from a 0.2:1 mixture of rXH(+)D(–) and rXH(–)D(–) prior to in vitro selection.

 
The results of selection with a mixed pool of mRNAs under conditions similar to those used for ribosome display (no stop codon in the ORF) are shown in lane 7, which shows that the dominantly selected mRNA from the 1:1 mixture of mRNA pools was the histidine tag-coding mRNA [+; Figure 4A (III)], demonstrating the appropriate selection of the target protein in the absence of a stop codon (as in the standard ribosome display system). We also investigated the selection with the initial pool originating from the 0.2:1 mixture of selection-positive and selection-negative mRNAs. As shown in Figure 5B, these results were in agreement with the results shown in Figure 5A.

Correlation between selection efficiency and the presence of a stable secondary structure at the 5' end of the mRNA

In the ribosome display, a stem and loop structure at the 5' end of mRNAs improved the efficiency of their selection system by reducing the rate of degradation of the mRNAs (Hanes and Plückthun, 1997Go; Schaffitzel et al., 1999Go). Since the Cv motif forms a characteristic secondary structure, we examined whether such a structure might also stabilize mRNAs and have a positive effect on selection. We predicted the thermal stability of the Cv motif (Figure 6A) and that of the stem–loop region used previously (Hanes and Plückthun, 1997Go) for stabilization of the ribosome–mRNA complex using the RNAstructure program Ver. 3.5 (Mathews et al., 1999Go). All the parameters used by the software were selected for standard free energies at 37°C. The free energies of the most thermostable structures derived from the Cv motif and the stem–loop region reported in the literature (Hanes and Plückthun, 1997Go) were –15.6 and –14.2 kcal/mol, respectively, indicating that these structures are similarly stable.



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Fig. 6. The most thermostable secondary structures of the Cv motif (A) as predicted by the RNAstructure program Ver. 3.5. (B) The results of in vitro selection, performed to estimate the contribution of the thermostable structure of Cv in the mRNA to the efficiency of in vitro selection. The patterns of products of RT–PCR after agarose gel electrophoresis are shown. Lanes 1–6 show products of RT–PCR derived from each mRNA pool after in vitro selection. Lane 1, the initial mRNA pool included only rCvH(+)D(+); lane 2, the initial mRNA pool included only rXH(+)D(–); lane 4, the initial mRNA pool included only rCvH(+)D(–); lane 5, the initial mRNA pool included only rXH(+)D(–). These lanes provided controls for identification of each product of RT–PCR by reference to respective mobilities during agarose gel electrophoresis. Lane 3, the initial mRNA pool was a 1:1 mixture of rCvH(+)D(+) and rXH(+)D(+); lane 6, the initial mRNA pool was a 1:1 mixture of rCvH(+)D(–) and rXH(+)D(–).

 
To estimate the contribution of the structure of Cv to the selection efficiency, we prepared plasmids with [pCvH(+)D] and without [pXH(+)D] the Cv motif that subsequently yielded four kinds of mRNAs, namely two mRNAs with and two without a stop codon. Each sequence, furnished with a T7 promoter, was amplified by PCR and the resulting products of PCR were transcribed to yield four kinds of mRNA: rXH(+)D(+) [Figure 1A (I)]; rXH(+)D(–) [Figure 1A (III)]; rCvH(+)D(+); rCvH(+)D(–). These four mRNAs were used for in vitro selection of the target (histidine tag-containing) protein.

All the mRNAs encoded a histidine tag and, therefore, all of them were selection-positive. However, if Cv were to contribute to the selection efficiency, we postulated that we would be able to collect increased amounts of Cv-containing mRNA [rCvH(+)D(+) or rCvH(+)D(–)] in the recovered pool of mRNA after selection from a 1:1 mixture of the Cv-containing and the Cv-non-containing mRNAs with Ni-NTA agarose beads. Lanes 1, 2, 4 and 5 show products of RT–PCR that originated from individual mRNA pools composed of each unique sequence (not mixed mRNA pools), corresponding to rCvH(+)D(+), rXH(+)D(+), rCvH(+)D(–) and rXH(+)D(–), respectively. These bands were used for reference.

The results of selection, using a mixed pool of mRNAs, are shown in lanes 3 and 6. Unexpectedly, analysis of the bands in lanes 3 and 6 demonstrated that the presence of Cv (upper band) did not improve the yield of the respective mRNAs either in the presence (lane 3) or in the absence (lane 6) of a stop codon. In both cases, the intensity of the upper band was equal to that of the lower band, and we detected no quantitative difference between the upper band and the lower band in lane 3 and in lane 6. Therefore, contrary to expectations, a stable structure at the 5' end of the mRNA did not enhance the efficiency of in vitro selection by, for example, stabilizing the mRNA against attack by RNases.

Contribution of the ribosome–mRNA complex for the yield of in vitro selection

To evaluate the relative contribution of the ribosome–mRNA interaction, we repeated the selections in the presence of 1 mM EDTA instead of 50 mM Mg(CH3COO)2. Under the low concentration of Mg ions, the ribosome–mRNA complex was disrupted but the Cv–mMSp interaction was intact. We prepared radiolabeled mRNAs using [{alpha}-32P]CTP and compared the radioactivity that remained on the Ni-NTA agarose beads just prior to the elution step. In this case, the yield even in the presence of a stop codon in the ORF was sufficiently high that products were detectable even when the binding buffer contained 1 mM EDTA as shown in Figure 7A.

This result suggested that the introduction of the mMSp–Cv interaction improved the stability of the mRNA–protein complex to a limited extent. However, the recovered mRNA consisted, unfortunately, of a 1:1 mixture of histidine tag mRNA (+) and histidine tag-lacking mRNA (–) (data not shown). Therefore, it appeared that the mMSp–Cv binding interaction did not exclude ‘shuffling’ in the absence of the formation of polysomes (in the presence of EDTA), namely replacement of the target mRNA with another unrelated mRNA in the complex of the nascent protein and its parent mRNA, even though the contribution of the mMSp–Cv interaction effectively enhanced the stability of the polysome complex in the absence of EDTA. Therefore, although the presence of the mMSp–Cv interaction is important for the selection, this interaction by itself is not sufficient to link the phenotype and genotype.

Estimation of the contribution of the mMSp–Cv interaction to the efficiency of in vitro selection

As noted above, the introduction of the mMSp–Cv interaction improved the stability of the mRNA–protein complex to a limited extent; only in the presence of this interaction could mRNA be recovered in the presence of EDTA, which destabilizes the ribosome–mRNA–protein complex. Finally, we prepared three kinds of radiolabeled mRNAs that were used for accurate estimation of the contribution of the mMSp–Cv interaction in the presence of 50 mM Mg(CH3COO)2, instead of 1 mM EDTA (Figure 7A). Two mRNAs [rCvH(+)mM2D(+) and rXH(+)mM2D(+)] included the histidine tag but differed in terms of the number of Cv. The last mRNA [rCvH(–)mM2D (+)] was a control that did not include a histidine tag and no recovery of this mRNA on Ni-NTA beads was expected.

The vertical scale in Figure 7B refers to yield, which was defined as the ratio of radioactivity associated with pelleted Ni-NTA agarose beads before the elution step to the radioactivity of the total initial input of radiolabeled mRNA. We carried out the same experiments twice, and observed the same trend of the results from two independent experiments. Figure 7B shows that the introduction of the mMSp–Cv interaction meaningfully improved our in vitro selection system.

Conclusions

The data presented in this report can best be explained in terms of the formation of polysome–mRNA–protein complexes. The linkage between genotype (mRNA) and phenotype (nascent protein) in our system is based on the fact that ribosomes are unable to release the associated mRNA after the protein encoded by the mRNA has been synthesized.

We were initially surprised to find that removal of the stop codon was not essential for successful selection in our system. Moreover, the introduction of the additional mMSp–Cv interaction improved the efficacy of the system (Figure 7B). The importance of the mMSp–Cv interaction in the determination of selectivity can be estimated from the data in Figures 2 and 5. We defined selectivity as the ratio of the amount of histidine tag-coding mRNA to that of histidine tag-non-coding mRNA, as estimated by RT–PCR, and we calculated that selectivity increased from 3 (without Cv; Figure 2A, lanes 3 and 7) to 6 (with Cv; Figure 5A, lanes 3 and 7). Therefore, the introduction of the mMSp–Cv interaction improved not only the yield of the target mRNA but also the selectivity of the protein in vitro selection.


    Acknowledgements
 
The gene for dihydrofolate reductase (dhfr) from E.coli was kindly provided by Dr M.Iwakura (AIST). We also thank Dr L.Nelson (AIST) and R.Wadhwa (AIST) for very helpful comments on the manuscript. This research was supported by grants from Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received June 20, 2003; revised October 2, 2003; accepted October 6, 2003





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