1651 CGSYWHPQC(2) CGTFIHPQC(3) HPQ 2 Phage display (common panning) 3 PMID:16258189 Streptavidin Biotin 1DF8,1LUQ,1MEP,1MK5,1N43,1N9M,1NDJ,1NQM,1STP,1SWD,1SWE,1SWG,1SWK,1SWN,1SWP,1SWR,1SWT,2F01,2GH7,2IZF,2IZG,2IZH,2IZI,2IZJ,2RTD,2RTE,2RTF,2RTG,2Y3F,3MG5, Not determined. Ph.D.-C7C phage display library (CX7C) 1652 SLMVSVFPFLWMDLVW(7) RRFPLYFLSELMELIL(5) SGTWAVFPFLWLDLMW(4) SLMMSVFPFLWMDLVW(1) SSMVSVFPFLWMDLVW(1) SLMVSVFPFLWMDLMW(1) CVSWLGFAWEWLQLLG(1) GSSFWAHWEKLLSESL(1) FAWHWKILSKFCLWLG(1) PRFPLYFLSELMELIL(1) RFYFLSALVLVLSGKL(1) LQVALFLVGLTRGLSE(1) VGFSDARALLFIVLEL(1) RLFIRARCKLLLLLRL(1) FLRLLARLFISCSVVI(1) FLCLLSLLHRSYFFGI(1) LCLIVCRRRLLVRVCF(1) LCICVVVRVLLWSSVQ(1) SMPFLFHWLNLYLHVC(1) GSAGYLWLLVMRSICV(1) F-x(3)-W-x(2)-L 20 mRNA display (common panning) 4 PMID:21423613 E3 ubiquitin-protein ligase Mdm2 Cellular tumor antigen p53 1YCR,4HFZ, Not determined. X16 mRNA display library The mRNA-displayed peptide library is incubated with MDM2 immobilized on beads through an affinity selection tag containing a ZZ domain and a TEV protease cleavage site, and unbound molecules are washed away. The bound molecules are eluted by cleavage with the TEV protease, and their mRNA portion is amplified by RT-PCR. The resulting DNA can be used for the next rounds of selection or analyzed by cloning and sequencing. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, authors performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. Authors identified an optimal peptide (PRFWEYWLRLME) named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. 1653 PRFWEYWLRLME(10) KSFQQYWQELML(9) KTFEEYWLMLMS(4) PSFWEHWVELML(4) KRFQDYWSELML(3) 5 mRNA display (common panning) 5 PMID:21423613 E3 ubiquitin-protein ligase Mdm2 Cellular tumor antigen p53 1YCR,4HFZ, Not determined. X(2)-F-X(3)-W-X(2)-L-X(2) mRNA display library ELISA In ELISA, MIP (PRFWEYWLRLME) could inhibit the MDM2-p53 interaction with an IC50 of 10 nM. Also MIP could inhibit the MDMX-p53 interaction at an IC50 of 120 nM. The mRNA-displayed peptide library is incubated with MDM2 immobilized on beads through an affinity selection tag containing a ZZ domain and a TEV protease cleavage site, and unbound molecules are washed away. The bound molecules are eluted by cleavage with the TEV protease, and their mRNA portion is amplified by RT-PCR. The resulting DNA can be used for the next rounds of selection or analyzed by cloning and sequencing. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, authors performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. Authors identified an optimal peptide (PRFWEYWLRLME) named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. 1654 INTLLSEINSILLDIISLL(13)[2.709 ± 0.168] INLLLMEINMILNEIITLL(3)[3.409 ± 0.061] INTLLMEINNILLNIIDFL(2)[3.088 ± 0.161] INTLLEEINIILNNIISFL(2)[3.079 ± 0.124] INSLLKEINNILISIIDFL(2)[2.709 ± 0.113] INSLLMEINTILNDIIMFL(1)[2.983 ± 0.185] ISSLLEEINTILMDIIEYL(1)[2.726 ± 0.079] 7 mRNA display (competitive panning) 10 PMID:21136209 Interleukin-6, IL-6 Interleukin-6 receptor 1P9M, Not determined. X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library ELISA In peptide ELISA, the absorbance at 490 nm was determined. Data were reproduced from the graph and expressed as the mean ± SD of triplicate determinations. The phage RNA(-) acted as negative control with A490 of 0.037. Beside, both RA07 (INTLLSEINSILLDIISLL) and CA11 (NQQLIEEIIQILHKIFEIL) peptides bound to GST-IL-6 in a dose-dependent manner and RA07 had higher affinity for GST-IL-6 (EC50=45.1 nM) than CA11 (EC50=142 nM). To determine whether CA11 and RA07 have inhibitory activity against IL-6, a KT-3 cell-based assay was performed. KT-3 cells are known to proliferate in response to IL-6. Results showed that RA07 suppressed KT-3 cell proliferation in a dose-dependent manner, but CA11 did not. The IC50 value of RA07 was 5.6 μM. Besides, the results of ELISA-based IL-6 inhibition assay suggested that RA07 inhibits gp130 from binding to the IL-6/IL-6R complex, but not IL-6R from binding to IL-6. The IC50 value of RA07 in this assay was 2.8 μM, which was almost the same as in the KT-3 cell-based assay (5.6 μM). After the beads were washed three times with selection buffer, 1-8 μM of IL-6 (Ajinomoto) was added and incubation continued another 2 h. The bound molecules were eluted with PreScission protease (GE healthcare). The most frequently occurring sequence, RA07 (INTLLSEINSILLDIISLL), bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130. 1655 INTLLSEINSILLDIISLL(6)[2.709 ± 0.168] INTLLMEINNILLNIIDFL(2)[3.088 ± 0.161] INSLLMEINTILNDIIMFL(2)[2.983 ± 0.185] ISSLLEEINTILMDIIEYL(2)[2.726 ± 0.079] INSLLKEINNILISIIDFL(1)[2.709 ± 0.113] 5 mRNA display (competitive panning) 10 PMID:21136209 Interleukin-6, IL-6 Interleukin-6 receptor 1P9M, Not determined. X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library ELISA In peptide ELISA, the absorbance at 490 nm was determined. Data were reproduced from the graph and expressed as the mean ± SD of triplicate determinations. The phage RNA(-) acted as negative control with A490 of 0.037. Beside, both RA07 (INTLLSEINSILLDIISLL) and CA11 (NQQLIEEIIQILHKIFEIL) peptides bound to GST-IL-6 in a dose-dependent manner and RA07 had higher affinity for GST-IL-6 (EC50=45.1 nM) than CA11 (EC50=142 nM). To determine whether CA11 and RA07 have inhibitory activity against IL-6, a KT-3 cell-based assay was performed. KT-3 cells are known to proliferate in response to IL-6. Results showed that RA07 suppressed KT-3 cell proliferation in a dose-dependent manner, but CA11 did not. The IC50 value of RA07 was 5.6 μM. Besides, the results of ELISA-based IL-6 inhibition assay suggested that RA07 inhibits gp130 from binding to the IL-6/IL-6R complex, but not IL-6R from binding to IL-6. The IC50 value of RA07 in this assay was 2.8 μM, which was almost the same as in the KT-3 cell-based assay (5.6 μM). The bound molecules were eluted with PreScission protease (GE healthcare). The most frequently occurring sequence, RA07 (INTLLSEINSILLDIISLL), bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130. 1656 INTLLSEINSILLDIISLL(2)[2.709 ± 0.168] INTLLMEINNILLNIIDFL(1)[3.088 ± 0.161] INSLLMEINTILNDIIMFL(1)[2.983 ± 0.185] INLLLMEINMILNEIITLL(1)[3.409 ± 0.061] 4 mRNA display (competitive panning) 8 PMID:21136209 Interleukin-6, IL-6 Interleukin-6 receptor 1P9M, Not determined. X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library ELISA In peptide ELISA, the absorbance at 490 nm was determined. Data were reproduced from the graph and expressed as the mean ± SD of triplicate determinations. The phage RNA(-) acted as negative control with A490 of 0.037. Beside, both RA07 (INTLLSEINSILLDIISLL) and CA11 (NQQLIEEIIQILHKIFEIL) peptides bound to GST-IL-6 in a dose-dependent manner and RA07 had higher affinity for GST-IL-6 (EC50=45.1 nM) than CA11 (EC50=142 nM). To determine whether CA11 and RA07 have inhibitory activity against IL-6, a KT-3 cell-based assay was performed. KT-3 cells are known to proliferate in response to IL-6. Results showed that RA07 suppressed KT-3 cell proliferation in a dose-dependent manner, but CA11 did not. The IC50 value of RA07 was 5.6 μM. Besides, the results of ELISA-based IL-6 inhibition assay suggested that RA07 inhibits gp130 from binding to the IL-6/IL-6R complex, but not IL-6R from binding to IL-6. The IC50 value of RA07 in this assay was 2.8 μM, which was almost the same as in the KT-3 cell-based assay (5.6 μM). After the beads were washed three times with selection buffer, 1–8 μM of IL-6 (Ajinomoto) was added and incubation continued another 2 h. The bound molecules were eluted with PreScission protease (GE healthcare). The most frequently occurring sequence, RA07 (INTLLSEINSILLDIISLL), bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130. 1657 INILIMEINNILKTIIREL(2)[1.252 ± 0.089] 1 mRNA display (competitive panning) 8 PMID:21136209 Interleukin-6, IL-6 Interleukin-6 receptor 1P9M, Not determined. X(3)-L-I-X-E-I-X(2)-I-L-X(2)-I-X(3)-L mRNA display library ELISA In peptide ELISA, the absorbance at 490 nm was determined. Data were reproduced from the graph and expressed as the mean ± SD of triplicate determinations. The phage RNA(-) acted as negative control with A490 of 0.037. The bound molecules were eluted with PreScission protease (GE healthcare). 1658 FPRWKLLAHWADRWWF(5) MHYEAVRVGRVLRFLA(4) IRMMVSFLRRQSRWWL(1) ARVFGLWLRSMSDMWL(1) MWVHSAAMATGLASRM(1) GNRWQYWIAFRMRYVA(1) RHLLLVDRLRAIALLL(1) RVLRYYLVGLALRQMA(1) AAVASMLRHLAGLFVL(1) FPRWKLLAHWADRGWC(1) FPRWKLLAHWADRWWV(1) FLRWKLLAHWADRWWF(1) SQRSTMYIAAVLRWLA(1) MVPSWVGLARVLRWLA(1) LVLLGRMLKFVA(1) 15 mRNA display (common panning) 5 PMID:20181936 B-cell lymphoma/leukemia 10 Not determined. Not determined. Not determined. X16 mRNA display library Fluorescence polarization To evaluate the affinity of the selected peptides for the BH3-recognition site of Bcl-XL, competition fluorescence polarization assay was performed. The selected peptide A10 (FPRWKLLAHWADRWWF) (IC50=0.9 μM) showed marked inhibitory activity. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them (FPRWKLLAHWADRWWF) has higher affinity (IC 50 = 0.9 μM) than Bak BH3 (IC 50 = 11.8 μM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. 1659 YQVARMLRRVADQMAS(7) KVVMRQLLMIADTMAR(1) LTVARRLKWVADIIFA(1) 3 mRNA display (common panning) 5 PMID:20181936 B-cell lymphoma/leukemia 10 Not determined. Not determined. Not determined. X(2)-V-X-R-X-L-X(4)-D-X-I-X(2) mRNA display library Fluorescence polarization To evaluate the affinity of the selected peptides for the BH3-recognition site of Bcl-XL, competition fluorescence polarization assay was performed. The selected peptide B01 (YQVARMLRRVADQMAS) (IC50= 6.4 μM) showed marked inhibitory activity. 1660 ALLSDLYKLL(1) DSASDLYKLL(1) ESDLFKMASS(1) FLYMSDLYKL(1) HTQFSDLFKL(1) LMHKARTTWP(1) LSDLSKLAQS(1) LSQNGLTSFN(1) MCNHSDLNKL(1) MWSDLTKLLP(2) NMSFSDMWKL(2) PEMSSDLWKL(1) PLYSDLLKLL(1) PQDSDLYKLS(1) RIWMSDLVKL(1) RLQDSAVSGH(2) SDLWKMMDKN(2) SHRMSDLWKL(1) SNYVSDLYKL(1) SVKAWPSAQI(1) SYTESDLHKL(1) TLRFSDLIKL(1) TSSDLWKLLP(2) TTHSIVSHVS(1) VYTSSDLNKL(1) WLVESDLYKL(2) YSDLDKMKPY(1) YSDLVKLSTL(1) x-S-D-L-x-K-L 28 mRNA display (subtractive panning) 4 PMID:18855146 Anti-p53 monoclonal antibody Cellular tumor antigen p53 Not determined. Not determined. X10 mRNA display library In order to remove non-specific binders to antibodies, the molecules prepared in the previous section were incubated with human normal IgG (Sigma)-bound Protein G-Sepharose beads (GE Healthcare) for 30 min at room temperature with rotation. Although no peptides that fully matched with hTP53 were found in the clones isolated, the core sequence was found in hTP53. A 10-amino acid peptide containing the core sequence was chemically synthesized to verify its binding with SPR. Its Kd value for the antibody was 6 nM. 1661 AEYSSDLWKL(1) DYSDLNKLMH(1) ERFSDLMKLA(1) FIDLYNLLMP(1) FPFMSDLYRL(1) FSDLDKLKAT(1) FSDLWKLSVVE(1) HGYSDLWKML(1) HNPFSDLHKM(1) KPKNSARLHQ(1) KTPYSDLWKL(1) LLESDLYKLL(1) LTFSYLWKLR(1) LWTFSDLNKL(1) LYSDLTKLQC(1) LYTSSDLWKL(1) MTTSDLWKLL(1) MYSDLDKLLI(1) NLHYNDLFKL(1) PELFSDLWKL(1) PFSDLDKLIP(1) PPSDLDKFNT(1) PYWSDLHKLA(1) QPLYSDLYKL(1) SDKLSDLHKL(1) STFSDLWKLA(1) SVYSDLHKLA(1) SYSDLYKLCT(1) SYYTDLDKLL(1) TSLSDLYKLS(1) YISDLWKLLF(1) YLWSDLWKLQ(1) YSDLNRLKNT(1) YSDLSKLLTL(1) x-S-D-L-x-K-L 34 mRNA display (subtractive panning) 5 PMID:18855146 Anti-p53 monoclonal antibody Cellular tumor antigen p53 Not determined. Not determined. X10 mRNA display library In order to remove non-specific binders to antibodies, the molecules prepared in the previous section were incubated with human normal IgG (Sigma)-bound Protein G-Sepharose beads (GE Healthcare) for 30 min at room temperature with rotation. Although no peptides that fully matched with hTP53 were found in the clones isolated, the core sequence was found in hTP53. A 10-amino acid peptide containing the core sequence was chemically synthesized to verify its binding with SPR. Its Kd value for the antibody was 6 nM. 1662 DATVCSRVWSSHC(1) ECARVVPGLMGYC(1) ECAVDVFVYMVSC(1) ECCEAYHLYLTPC(1) ECCGISRGVRTXC(1) ECCLWKSGHGSMC(1) ECDRRCKFKFRAC(1) ECGARGLECAGAC(1) ECGFLTEADCGSC(1) ECGHGSRGGPGCC(1) ECGSDGRELRYGC(1) ECKLHGGSCSCAC(1) ECLKKIEVSQLSC(1) ECQCRIFSKRSSC(1) ECRKPRGGYRDAC(1) ECRLLRGHGEARC(1) ECRRVPLAPQGSC(1) ECTLWDSREQPTC(1) ECVEGIDANPVAC(1) ECVLVAWFDVREC(1) ECWCIIRPVEPCC(1) ECWEDLVDDWRDC(1) GAGERFNVWC(1) GCAFARXLXKVFC(1) GCATKMVGAPRCC(1) GCCGGVRHTGFTC(1) GCCGSGFSLEEVC(1) GCDRGVELVNGVC(1) GCESVSRTPHAGC(1) GCFHVRMLTARGC(1) GCGAAREGGADRC(1) GCGGILKVWLVAC(1) GCGHGAAADALHC(1) GCIVALWLVFQIC(1) GCLGFDDQGIGRC(1) GCLSRVCVRYSAC(1) GCLVGDLGACAVC(2) GCMFCRSGDFASC(1) GCNVPTCQAPRRC(1) GCNWLILDAAFSC(1) GCQQEHGCHRYSC(1) GCRGGADYTTSYC(1) GCRNSGVSAGGKC(1) GCRSRSMLSMRMC(1) GCSAWADLELDTC(1) GCSRGKKKWVIRC(1) GCSTTDIQGLLYC(1) GCSVAGLLHAVGC(1) GCTALEGGRTAHC(1) GCTPRSLDSYGGC(1) GCTVGCRNAAEPC(1) GCVDPCTCVRPGC(1) GCVGGGTLSSLNC(1) GCVIEGRYFRCRC(1) GCVLAITRRSVWC(1) GCVLFVSTREYVC(1) GCVRAIVFGIYIC(1) GCWMAVGNHEVSC(1) GCWTDVSYWSFFC(1) GCYRWPVEKPLDC(1) KCALVFRSDGVLC(1) KCCRTLQHAVWRC(1) KCDGVECVAVEKC(1) KCFLTSGSWVSAC(1) KCGWNAIKSEGGC(1) KCHGCTCREWDHC(1) KCNFAVLSGDLVC(1) KCSRGIRCAGVLC(2) KCVRNLANGGAVC(1) KCWLCYGGLYNYC(1) KCWLSFAPYAGCC(1) RCATIRAVYAHSC(1) RCCLDVVFWQVPC(1) RCDASHALKKLHC(1) RCDGNRVVFAVIC(1) RCFSQASRDAEVC(1) RCGACGHVCRYTC(1) RCGAWQMLLLLGC(1) RCGCGRSSSLFNC(1) RCGSREGLSFLQC(1) RCIVNGPVTRILC(1) RCLQSRWDGFPWC(1) RCLRAGLPCSNSC(1) RCLSALTCKMALC(2) RCLTGTFAPWAFC(1) RCMLILRCYSAGC(1) RCRASGAGACMLC(1) RCRHRFLRFVASC(1) RCRLEPPDCRTRC(1) RCRLYDMLAVCLC(1) RCRQIGCALVGQC(1) RCRSSQTATASRC(1) RCRWGGVSGFFLC(1) RCSQIAVSIIGPC(1) RCSTRAEAAREGC(2) RCTCLGLHHFIKC(1) RCVGRVDKNKFLC(1) RCVGVIWDSEARC(1) RCVHSSTAVWFRC(1) RCWGTRMGQRGTC(1) 100 mRNA display (competitive panning) 6-7 PMID:18824687 Internal ribosomal entry site, IRES Not determined. Not determined. Not determined. [RKEDG]-C-X(10)-C mRNA display library 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. 1663 GCGSCPVCHGYPC(1) GCGGTEGHIGRGC(1) GCCWTAAMAGTSC(1) KCSRGIRCAGVLC(15) KCSRGIRCAGVRC(1) KCSRGIRCAGVLX(1) GCTACDRMYLVCC(1) GCRVCNRMEGVLC(1) GCLYLWRQGGLAC(1) RCIDHQYSWLCYC(1) ECGCSHPVFLCYC(1) RCWESYVDHLDLC(1) GCQSYGPGDLCLC(1) GCVGYSRIGLSVC(1) GCWISISACFKSC(1) GCVYSRLKVFADC(1) GCWVEMRGSWKRC(1) GCWGCMLSPEFRC(1) ECIASSGRAGGVC(1) GCRAASSVSWMWC(1) KCKKPGSARWSKC(1) RCNKSLIAIRYDC(1) ECGYRKIARMVMC(1) RCPSEGYHRRTGC(1) ECCQSGRPRDGGC(1) GCSGVGAARTWGC(1) GCTFGTQPRHWCC(1) ECHVTQHPYSRAC(1) KCKGFVGFFSRAC(1) ECTDCYLALSSYC(1) ECRRCFAELSYAC(1) ECLCYSYAGSCRC(1) KCTLFKAAGGPFC(1) RYGSFDDMSCGEC(1) GCYDRMPGGTHSC(1) GCGLGRARTDVAC(1) RCEGCVHYMGLSC(1) GCDRWSAGCVVC(1) RCAFCDFLTRXLC(1) 39 mRNA display (competitive panning) 11 PMID:18824687 Internal ribosomal entry site, IRES Not determined. Not determined. Not determined. [RKEDG]-C-X(10)-C mRNA display library 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. Authors 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 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. 1664 HCYPLDESILRLVRGELGAVH YWFAWAKSWQLAKREGHMGFD NWSGFYNLKLNTLKSVRQVHY NWHHALLSDIVSALNKSSLWN NCGTEMFCMDKINEWREVYVD NWSLIHKKYLQQAKMKYARCH DWTLMKVHVKQCVRKLESFTN HCRFVRRVAGLMFRERLEDDH NWRLSHNAVLDDARVQYGVHH 9 mRNA display (common panning) 12 PMID:17879061 Thymidylate synthase mRNA, TS Not determined. Not determined. Not determined. YAC-TGZ-(XYZ)18-YAC mRNA display library Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. 1665 NWENKSDVLNKMLHLRLITN(1) HWEKIRDNVIHKSDRSYYQKH(1) NWKSIIQRNLRWNKFKRFYQD(1) NWNILRQEVMKMGPAKDTVRN(1) NWNTVWSAYRRASSKLTKKTY(1) NWNRWRWIYLAQKNHKECLKN(1) YWKRYRLMALKANFGSRYSRN(1) NCPSTRKRWIVYLYRGRLINN(1) NWNRVRRYCKCKYTLVAKNNY(1) NWYTVKQMMIKDRRRSQAMYN(1) NWSDIRMHGLMRRREKNVLNH(1) NWVKLRQRVTLAKRVAVNLNY(1) HWNKVASERINKMRRKAILNY(1) HWNAIKNVVHSQRMTKKIAMN(1) NWEILRSKYNSHMTKSNVFTY(1) YWSELRRKKWWKVWKLSSCPH(1) YWNHELRVRLINRIKMTN(1) HWGIIRQKIVVAHDIFQCKDY(1) NWINNVRLRIHTKRWLLKSNH(1) NWLRLVPRIKALNKVQVKNHN(1) NCKMQPQNWYHVYRMSRLVKN(1) DCEYRDKVTLFNLVRLVMTKN(1) YWSNRVTQSIKARYVIDSWQD(1) NWHKVFIRRQSKKLVYNTIKN(1) YCHKYTVANESHWSKIRLKMY(1) YWVFLKKKMKLENKCVRVVKN(1) DWNMLKVKLYALRVRRRRMAN(1) HWMRTSQRVRVNNAFHKYMGY(1) NCEVLKTQRWRKVLQQHIIRH(1) HWSKTQGGNKRWRMIGAVVAH(1) NWDKVRSTFKKCHSIVIFKRN(1) YWSLIVSKLRRRKVMNDPSTY(1) HWDRVRLTMLRSRLKDDKKKH(1) NCLKQRLLRTPYLMMSRAVTH(2) NWRKAINLVRKWRNNDDPNKD(1) HWAMTRWHILANNVMNRRTCD(1) YWQRLQSMLKRVDPRPYRVRD(1) HWNKLSYTVRIGEIKRYVWRN(1) YWRQKAKDDLMVRRLRRAVKH(1) NWRNLRLKWRRLNSVHRH(1) 40 Phage display (common panning) 2 PMID:17685586 Calmodulin, CaM Not determined. Not determined. Not determined. X20 mRNA display library The mRNA display library passed through a precolumn with streptavidin beads to minimize the enrichment of matrix-binding sequences. The flow-through was incubated with biotinylated CaM. Molecules that bound to CaM in a Ca(2+)-dependent manner were eluted using the same buffer containing 2 mM EGTA. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca(2+)/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. 1666 MMFLRCRATKML MMLPCCRATKML MVCCACRATKML MSYCWCRATKML MSLVVCRATKML 5 mRNA display (common panning) 1 PMID:16122707 Botulinum neurotoxin A light chain Not determined. Not determined. Not determined. M-X(4)-CRATKML mRNA display library Purified biotin-ubiquitin BoNT/A LC immobilized on Softlink monomeric avidin (Promega) was first incubated with the RNA-peptides for specified time. RNA-peptide fusions bound to the immobilized BoNT/A LC were eluted off the column with 5 mM biotin in the same buffer. 1667 MVMLACRATKML MWMTSCRATKML MAFFGCRATKML MLLGGWCRATKML MVLVGCRATKML MFRLACRATKML MLTPRCRATKML MLVPACRATKML MGSAYCRATKML MGLLCCRATKML MGLRFCRATKML 8 mRNA display (common panning) 2 PMID:16122707 Botulinum neurotoxin A light chain Not determined. Not determined. Not determined. M-X(4)-CRATKML mRNA display library Purified biotin-ubiquitin BoNT/A LC immobilized on Softlink monomeric avidin (Promega) was first incubated with the RNA-peptides for specified time. RNA-peptide fusions bound to the immobilized BoNT/A LC were eluted off the column with 5 mM biotin in the same buffer. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. 1668 MVMLACRATKML(3) MWMTSCRATKML(1) MAFFGCRATKML(1) MPGARCRATKML(1) MLGQHCRATKML(1) MLRGFCRATKML(1) 6 mRNA display (common panning) 3 PMID:16122707 Botulinum neurotoxin A light chain Not determined. Not determined. Not determined. M-X(4)-CRATKML mRNA display library Purified biotin-ubiquitin BoNT/A LC immobilized on Softlink monomeric avidin (Promega) was first incubated with the RNA-peptides for specified time. RNA-peptide fusions bound to the immobilized BoNT/A LC were eluted off the column with 5 mM biotin in the same buffer. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. 1669 YRTNHHYDVGRFAARGRRD(1) NGRSSMNWRSQEITRYTSEHHYRMAFL(1) PEQYDHHHLEARRRASSTRQVRARARR(1) RAYTPHHHAEGRLVRLEPHPAPYKNRT(1) YYVKNRLHHHRLARLVAAEHAHRLRVQ(1) NKRNLSYPWSHHHQVARRTHMRAQHTM(1) RPTKNFEAEVVRSTGPMHHHDTAKQRY(1) DFLTYNKSMGGRPTNFRHHHSSVVQSQ(1) DEPEVVGRVLGERPAGALADHHHMMKW(1) EVLHGHHHVVARVRASCTGPTRRASCA(6) HVYEKANNRLGHKHHHLAARRRSKSWN(1) SNKGFSWRKKGMAVTPNRHLHHHMVAH(1) TNHRHHHGVLERRQDILTGSLIEHKH(1) ILKRLREQHRHHHAAAHHVRVRRRGRH(1) NYTTRRAEWNRQDAHRHHHQEARRGAL(3) SKKDNAVGLQELRLREGHRHHHDVMLT(1) KKVRGHHRHHHQVALLDAAERGPGRMS(1) GIHHHHAMAVLAELGMNPMGFALPDMW(1) AGVHHHHDAARGGTRSRRSTPRSATRR(1) TMNWHHHHENGLRARMYDAGRR(1) KVRRDVMRWHHHHRMARRKANR(4) RVQDRLGHRAVQPVLHHHHQAARRRVR(1) AALHHHHHDAGRASAMRRPGTPATSWR(1) DGHPERHDAGDHHHHHGVRQWRLISTG(20) 24 mRNA display (subtractive panning) 6 PMID:15980016 Anti-polyhistidine monoclonal antibody HIS-1 Recombinant polyHistidine tagged fusion protein Not determined. Not determined. X27 mRNA display library The reverse-transcribed fusions were pre-cleared by rotating with 20 ml of protein G-Sepharose for >1 h. Bound RNA-peptide fusions were eluted with acetic acid through a 0.45 mm spin filter (SpinX, Costar). After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined by constructing a high-complexity, unidirectional fragment library from the final selection pool. Selection by mRNA display minimized the dominant peptide from the original selection to a 15-residue functional sequence (peptide Cmin: RHDAGDHHHHHGVRQ; K(D) = 38 nM). 1670 MSQTKRLDDQLYWWEYL 1 mRNA display (subtractive panning) 6 PMID:15248784 Guanine nucleotide-binding protein G(i) subunit alpha-1 Not determined. Not determined. Not determined. GPR X(6) mRNA display library Surface plasmon resonance (SPR) The dominant peptide from the selection, R6A (MSQTKRLDDQLYWWEYL), retain high affinity (KD = 60 nM) for the GDP-bound state of GiR1, as measured by surface plasmon resonance. The affinity matrix for selection was prepared by rotating Nb- and/or Cb-GiR1(~10μg each) with ~20μL streptavidin agarose (Immobilized NeutrAvidin on Agarose, Pierce) in buffer A (20 mM HEPES-KOH at pH 7.5, 200 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween 20) at 4 °C for >1 h. The slurry was supplemented with 1 mMD-biotin (~0.1 mM final concentration) and rotated for an additional 10 min to block biotin-binding sites. After washing thoroughly with buffer A2 [buffer A supplemented with 2μM GDP, 1 mM β-mercaptoethanol, 0.2% (w/v) BSA, and 1μg/mL yeast tRNA (Roche Diagnostics Corp., Indianapolis, IN)], reverse-transcribed fusions were rotated with the affinity matrix in 1 mL of buffer A2 at 4°C for 1 h. A total of 6 rounds of selection were performed on a mixture of immobilized Nb- and Cb-G iR1 to reduce the effects of bias or steric hindrance with either terminus immobilized. Detergent, bovine serum albumin (BSA), and salt were included in selection buffers to minimize recovery of nonspecific binding peptides. DNA sequencing of the 6th round pool revealed a dominant peptide sequence (MSQTKRLDDQLYWWEYL). Selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A (MSQTKRLDDQLYWWEYL), and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(iR1), as measured by surface plasmon resonance. 1671 MSQTKRLDDQLYWWEYL MSQSERLDDQWTWWEFL MSQSKWLDDQLTWLEFL MSQSKRLDDQLTWLEFL MSQSKQLTITEFLQWL MSQSKRLEITWWEFVE 6 mRNA display (subtractive panning) 8 PMID:15248784 Guanine nucleotide-binding protein G(i) subunit alpha-1 Not determined. Not determined. Not determined. GPR X(6) mRNA display library Surface plasmon resonance (SPR) The dominant peptide from the selection, R6A (MSQTKRLDDQLYWWEYL), retain high affinity (KD = 60 nM) for the GDP-bound state of GiR1, as measured by surface plasmon resonance. The affinity matrix for selection was prepared by rotating Nb- and/or Cb-GiR1(~10μg each) with ~20μL streptavidin agarose (Immobilized NeutrAvidin on Agarose, Pierce) in buffer A (20 mM HEPES-KOH at pH 7.5, 200 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween 20) at 4 °C for >1 h. The slurry was supplemented with 1 mMD-biotin (~0.1 mM final concentration) and rotated for an additional 10 min to block biotin-binding sites. After washing thoroughly with buffer A2 [buffer A supplemented with 2μM GDP, 1 mM β-mercaptoethanol, 0.2% (w/v) BSA, and 1μg/mL yeast tRNA (Roche Diagnostics Corp., Indianapolis, IN)], reverse-transcribed fusions were rotated with the affinity matrix in 1 mL of buffer A2 at 4°C for 1 h. The first 6 rounds of selection were performed on a mixture of immobilized Nb- and Cb-G iR1 to reduce the effects of bias or steric hindrance with either terminus immobilized. Detergent, bovine serum albumin (BSA), and salt were included in selection buffers to minimize recovery of nonspecific binding peptides. An additional 2 rounds of selection was preceded by a subtractive hybridization step. Selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A (MSQTKRLDDQLYWWEYL), and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(iR1), as measured by surface plasmon resonance. 1672 MLRNSNCIRHFFGVDYKDDDDKGGGG(2) MDRTPTCTFLSSGGDYKDDDDKGGGG(1) MNFANVCVSQHIGGDYKDDDDKGGGG(1) MATKTDCFLSLVGGDYKDDDDKGGGG(1) MNARWNCNSWLVGGDYKDDDDKGGGG(1) MGRHTPCVSNLYGGDYKDDDDKGGGG(1) MSPYRSCGLSASGVDYKDDDDKGGGG(1) MRNNITCRLLKRGVDYKDDDDKGGGG(1) MRACTTCSWPLSGVDYKDDDDKGGGG(1) MRHNLNCSAFWPGVDYKDDDDKGGGG(1) MSLSSICLVPVAGATTRTTTTRAAA(1) MESHMVCRSTDVDYKDDDDKGGGG(1) MSPAHCCPYLPFDYKDDDDKGGGG(1) MSLAAHCAFPFLDYKDDDDKGGGG(1) 14 mRNA display (common panning) 9 PMID:12670537 Penicillin binding protein 2a, PBP2a Not determined. Not determined. Not determined. X(5)-C-X(5) mRNA display library A fixed cysteine residue in the mRNA fusion library (X(5)-Cys-X(5)) reacts with sodium 6-bromoacetyl penicillanate to form a peptide-drug conjugate. In vitro selection using this hybrid peptide-drug library resulted in novel inhibitors of the Staphylococcus aureus penicillin binding protein 2a (PBP2a). This strategy resulted in a penicillin-peptide conjugate (LRNSNC[Pen]IRHFF) that has at least 100-fold higher activity than the parent penicillin itself. 1673 ERNYNDFCDPGRVGL(8) YFCMGQICFLRFALH(2) DFDNMFSNLDPGRHW(2) FFDRYDSARDPGRLL(2) ERNYNDFCDPGRGGL(2) ERHNDPGRYFVEYET(2) FYESCENSDPGRTYA(1) FHESCENSDPGRTYA(1) YTHSSKSSDPGRKLW(1) DYSNESSDPGRLWHC(1) ERNYNDFCDPGRFGL(1) ERNYNDFCNPGRVGL(1) ERNYNDLCDPGRVGL(1) WRHYNPSDDPGRVHL(1) EYTNDYDDPGRTLSG(1) RNYTNPYCDPGRQHE(1) HGPDVNHADPGRYFD(1) LHLDSDHFDPGRTVW(1) LVDCISHDDPGRSVG(1) DINYCDPGRDCDGHL(1) DSYCELTDPGRWISA(1) EPCCCENRDVGRLIH(1) EFNQWEDPGRMRVGC(1) NHNLMNDPGRFFWHD(1) YTYSNDFTDGGRHIL(1) DYVSDVCRDGGRIML(1) YFDPGFCVFTSDHLA(1) YDAGFCNYDRDHIWP(1) YNSLSGRSLHPDIGF(1) DNYKLCESDVGRLLF(1) WFVYIDRWAYASFRH(1) YVYLLRHDQHSYYPP(1) YLHVVEIERHRIRFF(1) WEIYWHLEFAGFDRV(1) YALIYAVKKRMGIAH(1) WQRCGMAEFIWHGQW(1) YGHCFENFGDSFEHN(1) DYWAFWRVYFQVDGY(1) LYVRRSTAHIFVYAN(1) WLVCRHSKRYNCIFL(1) LRLVGSDHNFDAVVC(1) YMSFTRRTESDKLHS(1) DRSPRMFYNRFNSAL(1) LNWPNNLEGRDPYNR(1) DRSILVPRYFELWAN(1) DPGR 45 mRNA display (common panning) 10 PMID:12573700 Prothrombin Not determined. Not determined. Not determined. X15 mRNA display library Surface plasmon resonance (SPR) Thrombin was used as analyte at increasing concentrations (1-2500 nM). Dissociation constants were then calculated by the BIAevaluation software using steady-state affinity curve fits. Kd values of 520 ± 6 nM for T10-11 (ERNYNDFCDPGRVGL) and 166 ± 18 nM for T10-39 (FFDRYDSARDPGRLL) were determined. Bound peptides were eluted with elution buffer (4 M urea, 0.5% SDS). Two clones (FFDRYDSARDPGRLL and ERNYNDFCDPGRVGL) showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-meditated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. 1674 MDAQTRRRERRAMERAQTSSSL(1) MDAQTRRRERRAMERATLPQVL(11) MDAQTRRRERRAALRNEKFWVV(1) MDAQTRRRERRALMRRNARIAV(1) MDAQTRRRERRARDRFRLLRTL(1) MNAQTRRRERRALAFKARYMAL(1) MDAQTRRRERRAWERRTQSWMV(1) 7 mRNA display (common panning) 11 PMID:11675487 Three RNA targets (λ-boxBR, GNRA tetraloop, and P22 boxBL) Not determined. Not determined. Not determined. X10 mRNA display library Each peptide tested could bind at least one of the hairpins in the mixture. The specificity of seven individual clones, the round 11 and round 12 pools were then tested by using each individual hairpin. All showed a marked preference for the boxBR RNA as compared with the GNRA or P22 targets. 1675 MDAQTRRRERRAWKTVLWLQAL(1) MNAQTRRRERRAMEREERSKTV(1) MDAQTRRRERRALERTKLEKAL(1) MDAQTRRRERRALQRSRARHAL(1) MDAQTRRRERRAMERLMHERQA(1) MDAQTRRRERRAHERKISFTAL(1) MDAQTRRRERRAELRKSSLAFL(1) MDAQTRRRERRAWERTQRKNEY(2) MDAQTRRRERRALSRKMAIRIL(1) MDAQTRRRERRANIRRQSAWVL(1) MDAQTRRRERRALERQKSLRAL(1) MNAQTRRRERRAKDLKNTGMPF(1) MDSQTRRRERRAVRRESQYGSL(1) MDAQTRRRERRANMRMYRSLVI(1) MDAQTRRRERRAQSRMTNSWVL(1) MDAQTRRRERRATMRDSRSLAL(1) MDAQTRRRERRALERSMKLHAL(5) MDAQTRRRERRALDREMRGMVL(3) MNAQTRRRERRATLREERRVSW(3) 19 mRNA display (common panning) 12 PMID:11675487 Three RNA targets (λ-boxBR, GNRA tetraloop, and P22 boxBL) Not determined. Not determined. Not determined. X10 mRNA display library Each peptide tested could bind at least one of the hairpins in the mixture. The specificity of seven individual clones, the round 11 and round 12 pools were then tested by using each individual hairpin. All showed a marked preference for the boxBR RNA as compared with the GNRA or P22 targets.