Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. arn aiae Reyes Galinanes Sabrina sequencing of high-throughput detection via rapid motif provide binding display consensus cDNA and display mRNA PURE eoigicesnl omni eetyasfravreyo iceia n oeua rcdrs mainly Kr procedures, (Hammerling, molecular biology and synthetic biochemical in of particularly variety and versatility, a their for to years due recent in common increasingly becoming Recombinant motif consensus FLAG screening, sequence Introduction peptide system, PURE display, cDNA display, mRNA The studies future Keywords display. for mRNA useful for polypeptides. be display diverse enrichment will mRNA of system higher between space PURE slightly comparison available functional Furthermore, commercially with and by sequence motif performance binding. achieved explore for similar methods binding to display essential overall FLAG different residues in two consensus key of resulted that two revealing consistency indicates method the (DYKxxD) as display as residues well key cDNA appear four (K) as and the Lys DYKDDD of and one epitope (Y) lacking motifs FLAG Tyr a FLAG as the efficient core antibody of 10 and of M2 Enrichment x clean enrichment DYK(D/L/N)(L/Y/D/N/F)D. anti-FLAG 1.7 available enabling clear approximately commercially assay from showed the downstream Starting chose sequencing the we validation. proof-of-concept, to for a system molecule and As target translation conjugates screening. the cDNA-peptide sequence from and peptide components mRNA random stable of of carry-over preparation minimum the allow for for utility method its display validated PURE-based refined and a native developed We of expression as such recombinant The Abstract 2020 4, June 7 6 5 4 3 2 1 Ditzler Eto ela iiiigtepsil iiain htcrywrigwt iigcls(alo,Gn Hodgman, Gan, (Carlson, as machinery, cells living translation with and working reaction transcription carry the the that in as flexibility limitations allow such possible systems the production, These minimizing protein 2017). Tan, as for & well parameters Villarreal 2016; of Jewett, condition & Stark, Perez, 2012; AAAe eerhCenter Research Ames NASA JST INSERM Inc MOLCURE, Technology Technology of and Institute Science Tokyo Marine-Earth for Agency Japan Glasgow of University 4 aoh Tamaki Satoshi , 7 n oueFujishima Kosuke and , nvitro in nvitro in nvitro in rncito-rnlto T-L rcdrs lokona elfe ytm,have systems, cell-free as known also procedures, (TX-TL) transcription-translation rnlto ytmkona h UEsse a enue navreyo elfe experiments cell-free of variety a in used been has system PURE the as known system translation 4 sk Kobayashi Asaki , eeto.Orcnuaefrainecec xedd4% olwdb e uicto to purification gel by followed 40%, exceeded efficiency formation conjugate Our selection. 1 enovo de ues Kuruma Yutetsu , 3 rtisa ela aiu ipa ehd oslc o ucinlpolypeptides. functional for select to methods display various as well as proteins 5 y Mizuuchi Ryo , 2 a Fujimi Mai , 1 12 admsqecs on-yrudhigh-throughput round-by-round a sequences, random 6 3 ynRothschild Lynn , aaoYamazaki Masako , gr eet 09 Keasling, 2019; Jewett, & uger, ¨ 7 Mark , 4 Sumie , Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. opoiea vriwo eunedsrbto Fjmr ta. 02.Hne epromdround-by-round performed we Hence, 2012). and al., et methods, technology (Fujimori display distribution display with sequence (Roosild, along cDNA of antibody used overview recently M2 and an been anti-FLAG provide has against display sequencing to sequences Next-generation mRNA epitope 2006). 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RNA translation Seelig, & of PURE 2020; Shin degradation Seelig, the free 2005; (Hino & minimize ribonucleases Wilusz, Tong, help especially especially Cabezas-Perusse, & can conjugates, and chemicals ton, Wilusz, mRNA-protein proteases nuclease-free Sokoloski, and of of Anderson, inhibitors presence instability Opyrchal, RNase the relative 2008; to the due al., by systems, et limited translation is lysate-based display cell in mRNA Szostak, & of Ricardo, utility the (Josephson, The screening and functional of for mRNA screening environment Moreover, display, an specific 2003). a between mRNA suit Roberts, linkage 2014). of to & modified phenotype-genotype case Austin, easily (Takahashi, covalent the be puromycin a can In using 10 creating transfection. encodes to by it or (up achieved polypeptide transformation molecules is of of libraries efficiency number large the highest by indispensable the limited become methods, have screen not display such can the as display and Among phenotype 1997; ribosome evolution. to Szostak, directed genotype & couple for Roberts to 1997; tools Yanagawa, strategies & various Husimi, use Miyamoto-Sato, 2011)) (Nemoto, YamaguchiSeelig, display 2016; Yonezawa, mRNA Kubo, 1999; 2009), Yanagawa, & & (Naimuddin & al., display Doi Cherf et cDNA 2004; 2003), 1997; Neri, Yanagawa, & & Higashinakagawa, Wittrup, (Bertschinger Kawahashi, display Doi, & DNA Pl 2013), (Boder Yomo, & (Ledsgaard, & Amstutz, display display Kazuta, 2018; (Zahnd, Sunami, (phage yeast display Tan, methods 2018), ribosome display & 2015), Laustsen, Different (Contreras-Llano Cochran, 2014). & al., McCafferty, peptide/proteins et Karatt-Vellatt, Fujii of Kilstrup, 2015; evolution conditions Sarkar, over & directed control system. Markou, a and needs PURE Dodevski, one screening the when in beneficial high-throughput 2019) particularly for are al., systems et recombinant contaminant-free (Wang of advantages a source 2019) more These energy Maerkl, Lately, offer & the 2014). (Lavickova 2014), Yomo, reconstruction altering for & Ueda, as low-cost Ichihashi, well and & Matsuura, robustness 2015; as Kanamori, simplification, (Kazuta, Ueda, on yield Kuruma, & focused protein has (Kuruma Shimizu, significant research system Yoshihiro final PURE a 2001; as with al., such alternative systems et continues ribosomes cell-free research purified Shimizu 2016), ongoing Doi, and Y While & proteases), Fujiwara elements lysate. (nucleases, 2017; protein Hasty, cell proteins & recombinant the Tsimring, and using Tonooka, in mRNA (Didovyk, included issues the factors these some unknown degrade mitigate however, additional to actively products; of that protein presence of components 2020; yield the Jewett, to high and & due produce Kay remain often 2020; systems al., still encountered These et issues constraints (Bowie 2019). exhibits Pardee, compound that tackled toxic & protein it growth of Jaenes, under as expression Tinafar, Sam-Yellowe, as production popularity such & protein Yadavalli organisms, gained or 2014; living toxicity quickly Zhao, using cellular & lysates research Zhao, biology cell Wang, synthetic living Suzuki, 2013; using performing Ezure, of al., when of 2014; variety et Schillberg, application Sun a & 1983; The proteins. Spiegel, from Hunt, Vogel, 2015). recombinant & extracts Buntru, cell- Jackson 1983; purified employ 2014; of Dudock, and use Ando, & types & Straus, developed, that main (Anderson, be those line Two to cell and HeLa 2019). first extracts Jewett, as, the cell such were & from organisms systems Karim, derive lysate-based Silverman, that cell 2012; those The Jewett, exist: & systems Hodgman free 2012; Jewett, & shrci coli Escherichia es,fl ryom ha em oac,rbi eiuoye and reticulocytes rabbit tobacco, germ, wheat armyworm, fall yeast, , nvitro in ipa ehd oepplrfrsreigantibodies screening for popular more methods display 2 ctu,20) iooedsly(ui,Matsuura, (Fujii, display liposome 2007), uckthun, ¨ nvitro in prahssc smN,cN,and cDNA, mRNA, as such approaches 13 ob etdbcuete are they because tested be to ) nvitro in reactions Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. gi aeul icre,adteple a i-re tro eprtr o 0mn ial,tepellet the was Finally, supernatant to min. 2000c. Two The added 30 Nanodrop min. was a for 15 ethanol by temperature for 70% quantified vitro room g of and In at x water ml 20,000 air-dried free 1 at , was RNase centrifuged and 4 in pellet After then discarded, at resuspended the mixture carefully performed was and was the to added. was discarded, supernatant and centrifugation was min carefully The second yield, Chemical) 1 again A the min. 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Gold Trypsin of g 2 n 7 MKCl) mM 375 and 2 7 mM 375 , Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. rne,20)bsdo h o 0raswt pcfi odtos(xdlte n positions). and letter (fixed & conditions Chandonia, specific Hon, with (Crooks, based reads 3 sequences 50 the WebLogo Results top using sort Only were created the rank format) were random on FASTQ to logos based the script. 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(GE using Reagent Selection Detection Blotting Western Select ECL using TC- n TC- rmr SplmnayTbe1.Terato i a nuae t8 for 80 at incubated was mix reaction The 1). Table (Supplementary primers RTPCR-R and RTPCR-F M μ μ frsseddmgei ed ihbudmN- RAcN-etd ojgtswr used were conjugates mRNA/cDNA-peptide mRNA-, bound with beads magnetic resuspended of l fAt-LGM antcBas(im-lrc)wr ahdtotmswt B- ue (50 buffer TBS-T with times two washed were (Sigma-Aldrich) Beads Magnetic M2 Anti-FLAG of l μ TPRrato oueuigPiecitOeSe TPRKtVr2wt 0.4 with Ver.2 Kit RT-PCR Step One PrimeScript using volume reaction RT-PCR l μ B ue n ouin eedrcl ple oteR-C ecin(e next (see reaction RT-PCR the to applied directly were solutions and buffer TBS l μ /lt 20 to g/ml μ /lt 100 to g/ml 5 μ fTSTbffr iue ape eemxdwith mixed were samples Diluted buffer. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. 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(Figure 40% above to rate formation the increased greatly KCl mM 375 and eeto n RT-PCR and selection 2 ocnrto,adicbto ieatrsl diin rnlto ierflcsthe reflects time Translation addition. salt after time incubation and concentration, ) 7 Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. PR ytm o ffiin n tbemN n RAcN-etd ojgt ytei n down- and synthesis conjugate mRNA/cDNA-peptide system and translation mRNA cell-free stable the utilizing and methods efficient display for cDNA and system) mRNA (PURE refined and established have 2003) We al., et 2006). Yonezawa al., 2013; Yamabhai, et & indicating (Roosild Srila Discussion round data result 1994; observed first structural al., The the antibody et antibody. M2 in (Miceli M2 obtained anti-FLAG enrichment selection the and previous negative of the showed recognition Enrichment with residues (DYKxxD). primary consistent motif for (K) is FLAG residues Lys core these or the of of importance (Y) through residues the evaluated Tyr four was lacking the binding with of sequences antibody one on than of lacking residues sequences greater acid of amino consistently comparison FLAG was individual the the elution of influence the the Finally, of 100 all among 1, after motif Table FLAG beads and the the conjugate of 4 to abundance display (Figure cDNA the bound display display over display, remain round) mRNA cDNA mRNA per condition, that with fold Additionally, selection sequences 28.7 round). our (average the motif per Under FLAG fold consensus 1). 19.1 the (Table eluant(average of respectively the rate display, enrichment from 100 cDNA higher recovered or shows and sequences (20 mRNA from peptide achieved for FLAG was RPM competitor of motifs concentrations FLAG high consensus with of sequences enrichment 4D). by highest replaced and The located were 4C DYK(D/L/N)(D/F/L/N/Y)D sequences (Figure selected motif: positions FLAG the different 4th consensus two of increasing final harboring at majority by the library The (DYKDDDDK) that For 10aa-random octapeptide manner. 4B). suggesting from stepwise epitope (Figure derived million, FLAG a antibody per the at in M2 reads with concentration residue anti-FLAG elution 31,689 the (D) the competitive and Asp to performed fixed 261,982 binding we with enhances of round, significantly (DYKDDDxx) enrichment respectively is motif marked display, cDNA FLAG position observed and consensus 6th we mRNA the round position, continu- 3rd on observed 6th the from we focused for sequence the progressed, we million epitope rounds per When the the reads detect 4A). as 198 to initial Although (Figure and able the 1479 sequences. not to in 1,768,526 were up of (DYKDDDDK) we enrichment sample that indeed sequence ous our and with epitope in million, display FLAG round per 0th mRNA full reads the the through 0.29 is for detected round) frequency motifs (0th FLAG expected library by various The followed in of display. probability rate features cDNA 80% enrichment of found over the further with compared 3D). we next (D) and and We motif, Asp 3C (D/L/N) and (Figure FLAG motif, et FLAG residue (K) Yonezawa core the (P) Lys 2013; the before Pro conserved Yamabhai, immediately with downstream the & (E) sequences between Srila Glu conserved those located 2009; weakly only (D/F/L/N/Y) al., (Figure as et such 5 on residues Osada position focusing variable at 1994; other By starting previous al., case the et in 2003). other (Miceli in (DYKxxD) the al., selection, antibody reported in motif of and M2 been FLAG rounds 2 anti-FLAG has core three position for motif conserved After at binding a 3A). starting 7th This case (Figure on charged 3B). one position converge positively in 8th library by variable positions, (D) followed 10aa-random different a Asp selection, two the and fixed of (K) from round a Lys derived first in and sequences the epitope resulted (R) FLAG after (DYKDDxxx) Arg of immediately top with library enrichment the position position the FLAG-random on 6th confirm focused the the first to from We at display Sequences residue reads. mRNA to of of script selection. number round their Perl the each et on during from a (Alam based software sequences sequences through FastAptamer abundant unique the parsed the most by rank 50 and analyzed and further count system were to Miseq sequences 2015) Extracted al., methods. the display region. from coding both 20 collected the for was extract for round data out 3rd sequence the carried for High-throughput was cycles PCR 16 and analysis reaction. libraries sequencing weak RT-PCR round remained High-throughput the 2nd band and with this 1st however proceeded bp, the we 200 for around thus cycles appearing rounds, band multiple non-specific a throughout observed also We selection. smr edl ltdfr h beads. the form eluted readily more is μ /lv ed) hs eut ugs httemRNA/cDNA the that suggest results These beads). vs g/ml 8 nvitro in μ /l ecigttl3597ad135,913 and 325,917 total reaching g/ml) eeto tde ohv togaffinity strong have to studies selection Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. ahrdi,N . ua .(05.Cntuto n cenn fvs irre fntrlproduct- natural of libraries technologies. vast of display screening vitro and protein-synthesizing for in Construction cell-free using a toolkit (2015). of https://doi.org/10.1016/j.cbpa.2014.11.011 peptides H. bioinformatic Preparation Suga, macrocyclic A & (1983). like S. K., FASTAptamer: B. N. Dudock, Bashiruddin, & (2015). germ. W., wheat H. J. from Straus, system D. W., Burke, C. Anderson, selections. & combinatorial L., of J. https://doi.org/10.1038/mtna.2015.4 analysis Chang, sequence K., high-throughput K. Alam, interest. References of conflict no declare authors manuscript. The the on comments valuable Interests and of their Fried of Stephen for detection Conflicts and University Technology, rapid Hopkins of Institute Johns allow Tokyo from of to Watanabe Lanning Hidenori Bryan targets and Nishikawa molecular Shota thank of We range broad to Acknowledgements applied Hence, quantitative 2019). be and Greenleaf, can high-throughput motifs. & binding McMahon, based method high-affinity (Layton, consensus array this variants the of explore epitope expect high-avidity further advancement important discover we to recent residues to useful influential able the be most assay with would the protein patterns along highlight Such of to candidates, pattern able recognition. enrichment epitope were antibody the we comparing M2 to difference, By anti-FLAG residue exist for residues. single important acid may contains a dependent amino also with residues database individual library sequence sequences of also a between large fitness We resulting in the epistasis The motif regarding round. high-order conformation. information FLAG final binding the suggesting optimal and the to 4th 10aa-random) for FLAG adjacent account the vs the acids by amino with variants (FLAG-random several sequence elution manner of other competitive enrichment over stepwise additional motif and variousobserved FLAG sequencing for consensus round-by-round mRNA/cDNA- revealed approach that larger peptide powerful show the We a the RNA-conjugated than on become the experiments. binding beads has non-specific because the more sequencing have possibly on High-throughput to display, sites appears cDNA binding library display more of cDNA beads. to that the remaining contrast, access than In it beads conjugates. with allows sequences peptide the but motif size FLAG method from display smaller our mRNA elute end peptides of The C-terminal to display. consistency the cDNA showing slower over to display performance, were close mRNA similar for too overall enrichment is motif. higher in FLAG one slightly resulted the sites, degenerate methods of three had display part library those Two original be of motif the to and the in region tyrosine, accommodate sites random for to three the code Only able of can were sequences; design. that motif that library FLAG sequences sites the consensus by the codon led the imposed of This of restrictions both discovery sequencing. the the in high-throughput within in appeared DYKD- with resulting motif identified epitope: residues, This was FLAG variable DYK(DLN)(DFLNY)D. canonical further, the was the into and pu- insights of methods selection enrichment new beads display of rapid to affinity two rounds A three the to 2006). after of al., comparison DDxx et Performance in (Roosild system 10 Also antibody approximately tags. PURE M2 from the affinity search anti-FLAG proteins. in epitope peptide present an and require conducting mRNA/cDNA- molecules over by RNA not the validated and carry target related do mRNA preventing to translation of polypeptides of intends capability and purity one rification, the if nucleotides the useful is as ensure be method such to could our steps This of electroelution conjugates. highlights peptide separate the two of One through components 1). (Figure selection stream ehd nEnzymology in Methods https://doi.org/10.1016/0076-6879(83)01044-7 . 9 12 urn pno nCeia Biology Chemical in Opinion Current etd eune gis h well-known the against sequences peptide oeua hrp uli Acids Nucleic - Therapy Molecular nvitro in eeto n evolution and selection . . Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. fcl-repoensnhssbsdo rd eletatfo shrci oi ha em n rabbit and germ, wheat coli, Escherichia from extract cell crude Efficiency a (2008). Y. on Baba, & based Y., Shinohara, synthesis reticulocytes. I., J. protein Kido, cell-free T., Yamamoto, of K., Kajimoto, M., Kataoka, M., Hino, machinery. without Kr synthesis protein J., cell-free M. for Hammerling, extract cell of preparation (2012). Biochemical E. disruption. of (2016). physical Miyamoto-Sato, production N. Doi, high-throughput . & dis- . for K., . Fujiwara, technology Liposome Y., Fukui, display A., cell-free data. (2014). Nishikimi, a interactome K., with T. reliable Masuoka, coupled Yomo, H., sequencing proteins. Ohashi, & Next-generation membrane N., Y., Hirai, of S., Kazuta, evolution Fujimori, T., and Nishikawa, selection T., of vitro https://doi.org/10.1038/nprot.2014.107 evolution Sunami, in vitro for T., In play Matsuura, (2013). S., T. Fujii, Yomo, & Y., Kazuta, https://doi.org/10.1073/pnas.1314585110 T., display. cells. liposome Sunami, insect a T., from using system Matsuura, synthesis S., protein cell-free Fujii, A (2014). E. Ando, Biology & Molecular T., Suzuki, protein T., combinatorial Ezure, of screening cell-free for in system fusion advances vitro. Protein-DNA in STABLE: methodological libraries and (1999). H. Conceptual Yanagawa, & N., (2015). Doi, A. Bacterial C. of Sarkar, Preparation & Scalable evolution. C., and directed G. Rapid Markou, I., (2017). generator. Dodevski, J. logo Hasty, Expression. sequence & Gene A Cell-Free L., WebLogo: for Tsimring, (2004). Lysates T., E. Tonooka, S. A., Brenner, Didovyk, & M., gene J. cell-free Chandonia, Research using G., Genome biomolecules Hon, of E., G. screening Crooks, High-throughput (2018). C. Tan, systems. expression & engineering. 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T7 the DNA harboring with the sequences) ligation to (randomized system. complementary library PUREfrex is DNA using that display designed cDNA a and with mRNA of starts overview Schematic 1. Figure sequencing and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high-throughput- image1.emf file Hosted vitro in proteins evolving and Selecting Figures target. display: a Ribosome to in (2007). bind for A. specifically display Pluckthun, that & DNA P., Amstutz, (2003). C., H. Zahnd, Yanagawa, (2009). libraries. & N. peptide T., diverse Nemoto, Higashinakagawa, of Y., selection and Kawahashi, . vitro . synthesis N., . solid-phase Doi, T., by M., peptides Yonezawa, Kubo, disulfide-rich M., functional fusions. Machida, for mRNA-protein method T., of screening Sasaki, stabilization novel M., A Biyani, Plasmod- display: of cDNA M., expression Naimuddin, for J., systems Yamaguchi, expression free cell translation based vitro proteins. Hela in rhoptry for (2015). ium system T. lysate Sam-Yellowe, cell-free & yeast R., Yadavalli, optimized An (2014). mRNA. N. virus K. human Zhao, E. of & S. L., McGlynn, Re- Zhao, and X., . Triphosphate . Wang, . Nucleoside N., of A. Regeneration Khusnutdinova, the Synthesis. Z., Driving Protein T. Cell-Free Kinase Jia, constituted Polyphosphate Y., Bifunctional Kuruma, A S., Berhanu, (2019). biology. K., synthetic Fujishima, of H., age P. new Wang, the in in ribosomes systems yeast Engineering Cell-free by and (2017). mediated Science C. reaction Tan, puromycin & The F., (1973). Villarreal, J. P. H. Bloemers, salt. & high A., C. Mast, Der Van oeua ilg Reports Biology Molecular vial at available at available e mgso h RAppiecnuaefrainudrvrostasainconditions. translation various under formation conjugate mRNA-peptide the of images Gel ora fVsaie Experiments Visualized of Journal ehd nMlclrBiology Molecular in Methods https://authorea.com/users/329657/articles/456613-pure-mrna-display- https://authorea.com/users/329657/articles/456613-pure-mrna-display- https://doi.org/10.1007/s11705-017-1610-x . 2 sdi h rvossuis(amdi ta. 01 amdi Kubo, & Naimuddin 2011; al., et (Naimuddin studies previous the in used aueMethods Nature https://doi.org/10.1007/BF00357646 . uli cd Research Acids Nucleic C ytei Biology Synthetic ACS uli cd Research Acids Nucleic https://doi.org/10.1038/nmeth1003 . 13 https://doi.org/10.1007/978-1-62703-782-2 . https://doi.org/10.3791/52772 . nvitro in https://doi.org/10.1021/acssynbio.9b00456 . https://doi.org/10.1093/nar/gkp514 . rncie omN irr n undergo and library mRNA to transcribed https://doi.org/10.1093/nar/gng119 . rnir fChemical of Frontiers h system The 14 Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. edcut o RAdslymto oag)adcN ipa dr le r rsne side-by-side. presented are blue) (dark 20, display 4, cDNA and at (orange) tag method epitope display FLAG round, mRNA each with for For counts elution sequences. read competitive xxxxDYK(D/L/N)(D/F/L/N/Y)D with (D) round and xDYK(D/L/N)(D/F/L/N/Y)Dxxx 4th selection, motifs. round FLAG enriched 100 3rd the to for (0th) counts library read Round-by-round 4. 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Data may be preliminary. bu) gen n eodD(akrd r hw o ahruddrn h A RAdslyand display mRNA (A) the during round each for shown are red) (dark core selection. D the display second from cDNA and residue (B) key (green) single K a (blue), lacking Y motifs the motif. of FLAG enrichment Round-by-round 5. 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Data may be preliminary. (100 (100 A B C Round 2 Round 1 Round 0 Round 4 Round 3 Conjugate Conjugate Conjugate formation formation formation μ

g/ml) (%) (%) (%) 10 30 50 10 30 50 10 30 50 ➖ ➖ 0 0 A probability probability probability probability probability 0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 FLAG-random FLAG-random 1/8 (DYKDDxxx) D D D D D 5 10 H N S Y Y Y Y Y A E R K K K K K A T 15 1/4 I 30 D D D D D C V A R D D D D D A T WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 5 5 5 5 5 E R D D D D D N K G S A 1/2 30 F E E E E E 60 R D R R R R D K K K K K N N N N N G G G G G Q S S S S S A E E E E E R R R R R D D D D D K K K K N N N N N G G G G G S S S S S V Time Salt Time 60 1 90 B (min)

probability probability probability probability probability (min) 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 I I I I L L E E R D P P R D H K N N K L E E E M R P P R P R D D D H H K N N N Q Q G G S S M M M V A A V T T G Q Q Q S V A V A V T T T 10aa-random 10aa-random (xxxxxxxxxx) I I I I I L L E E P R P R D H K N K N H E M R P R D D D N N H K H G G Q Q S A A V T T Q S A A T T T I I I L E F F R R P P D D L H N N L H F F F R P D D N H C N G G S S Y Y A V A T T C G S Y Y Y V A T T E I I mRNA/cDNA-peptide D L L E E P R D P R D N H K K L L L H N E M P P D R N H K K K G G Q Q S M V A V A T T G Q Q S Y V A T T I I I I I L L E E P P R R D D H N K L L H K N M E P M R D D D K H N K N N G G Q Q S S M M M V A A T T G Q Q Q S V A T T 5 5 5 5 5 I I L L F P P R R D D H N N H L L E F F P R D D D K H N N H N H G G S S Y Y V A A T T C C G S S S Y Y Y A T T T I I I L L E E P P R R D N H K H L K N M M R P D D D H K N H K K G G Q S S V A A T T G Q S V A T mRNA-peptide WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 16 I I I I L L E E P P R R D H N N H K K L L L E P R P P D D R D K N N K H N G G Q Q S S A A V V T T Q G Q Q S V V A T T T I I I I I mRNA-tag L L F P P R D R D N H H N L L L F F F P D P R P D D N C H H N C N H G G S S Y Y V A A V T T C C G G S S S Y Y Y V V A T T T conjugate I I I I I conjugate L L E E P P R R D K L H K N N L L H E R P M P P R D D D H mRNA-peptide K K H N N H G Q G Q S S M V A A V T T G G W Q S S S V A V A V A T T T 10 10 10 10 10 Tripsin digestion Translation mRNA-tag conjugate

probability probability C 0.0 0.5 1.0 0.0 0.5 1.0 probability probability 0.0 0.5 1.0 0.0 0.5 1.0 Translation I I L E E R P R P D D H N N H M M Q Q V V A T T (xDYKxxDxxx) I I 10aa-random 10aa-random L E E P P R R D D K H H K N N M M G Q Q S S V A V A T T D D RT D D Y Y Y Y K K N.A. K K I I L L D D N N M M Q Q V T ➖ ➖ I 5 5 I L L E E R R D D K N N M M G Q G Q V A V T T 5 5 L L F F D D H N N H C S S Y Y T T I L L F P R R D D H N H N C C G G Q S S Y Y V V A A T T D D WebLogo 3.7.4 WebLogo 3.7.4 D D WebLogo 3.7.4 WebLogo 3.7.4 I I L L E R P P K Q Q A V T T ➖ ➖ I I L L E E P P R D R N H K N H K ➖ ➕ G G Q Q S S V V T T FITC I I L F F R P P D D N N H H C S S Y Y V T T I I L L F F P R P D D N N H H C C G S S Y Y V A V A T T I I L L E R R P P D D K H N N H G S S A V V A T T 10 10 I I L L E E P D P R R D H K N N H K M M G G Q Q S S V A V A T T 10 10 ➕ ➖ ➕ ➕

probability probability probability probability D 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 0.0 0.5 1.0 ➕ ➖ Chemiluminescence I I I I L L E E E E P P P R R D R R P D D D H H K K K N N N H K G G G Q Q G Q Q S S S V V V A A A A V T T T T ➖ ➖ (xxxxDYKxxD) 10aa-random 10aa-random I I I I L L L L E E E E R P P P R P D R D D D K K N H H K N N N H H M G G Q Q Q S S S S V V V A A A A T T T T I I I I L L L L F F F P F D R P D D D N N N H H H N H C C C G G S S S S Y Y Y Y V V V A A T T T T I I I I L L L L E E E E R P P P R D R P R D D H K K N N H H K K N H N M M M M Q Q Q G G Q S V A A A V V V N.A. T T T T ➖ ➕ ➕ ➕ D D D D 5 5 5 5 Y Y Y Y K K K K WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 WebLogo 3.7.4 ➕ ➕ I I I I L L L L E E P R R D D D D H N H K N N N M M M M G Q Q S S V V A V A T T T T I L L L L F F F F P P R R D D D D H N N N H N H C G G G G S S S S Y Y Y Y A A V A A T T D D D D 10 10 10 10 Posted on Authorea 4 Jun 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.159129081.14061266 — This a preprint and has not been peer reviewed. Data may be preliminary. throughput-sequencing display-and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high- Table1-final.docx file Hosted vial at available https://authorea.com/users/329657/articles/456613-pure-mrna- 17