The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10 Specifications tableValue of the data•The finding that PoBChE is abundant in porcine milk •The set of polyproline-rich peptides in PoBChE tetramers is different from the set in human BChE tetramers •Are excess polyproline-rich peptides toxic to cells? Does BChE incorporate polyproline peptides from degraded cells because degraded cells are a convenient source? If excess polyproline-rich peptides are toxic to cells, BChE might be protecting the organism by scavenging these peptides.•The BChE tetramer incorporates not only short polyproline-rich peptides, but also long proteins that contain a polyproline-rich region. An example is the C5 variant of human BChE whose tetrameric structure includes a 60 kDa lamellipodin protein •The ability of BChE subunits to assemble into stable, long-lived tetramers by binding the polyproline-rich region of a protein, suggests that BChE could serve as a delivery vehicle for any protein that has been engineered to include a polyproline-rich tag.1We present all the polyproline-rich peptides associated with PoBChE tetramers that were identified by mass spectrometry. In addition we show the complete amino acid sequence of the full-length protein that donated each polyproline-rich peptide, the location of the polyproline-rich peptide within the full-length protein sequence, and the abundance of the polyproline-rich peptide relative to PoBChE peptides. A brief description is given of the function of the donor protein and its location within a cell.22.1PoBChE was purified from defatted porcine milk (1200 mL) by affinity chromatography on procainamide-Sepharose 2.2The Hupresin-purified PoBChE was reduced in volume from 7.3 mL to 0.17 mL in a Centricon YM-30 spin filter. After the buffer was changed to 10 mM ammonium bicarbonate pH 8, the PoBChE was deglycosylated with 1 µl of PNGaseF for 1 h. Noncovalently bound polyproline-rich peptides were released from PoBChE by denaturing the protein in a boiling water bath for 5 min. The denatured 170 µg of PoBChE protein in 170 µL of 10 mM ammonium bicarbonate pH 8 was digested with 2 µg of trypsin  for 20 h at 37 °C in a humidified chamber. Particles that could clog the small diameter tubing in the Ultra High Pressure Liquid Chromatography column were removed by centrifuging the digest for 30 min at 14,000 rpm in a microfuge. A 10 µL aliquot from the top of the centrifuged digest was transferred to an autosampler vial. The protein concentration in the digest was estimated at 1 µg/µL.2.3Sus Scrofa proteins using the Paragon algorithm from Protein Pilot (AB Sciex) (The protocol for liquid chromatography tandem mass spectrometry (LC–MS/MS) is described in detail in B Sciex) .Table 1AImage 1VSAKPPQALSFAKRLQQLIEALKGTAFSSGRSYYETETTDLQELPAS.>sp|P08001|ACRO_PIG Acrosin OS=Sus scrofa GN=ACR PE=1 SV=5 MLPTAVLLVLAVSVAARDNATCDGPCGLRFRQKLESGMRVVGGMSAEPGAWPWMVSLQIFMYHNNRRYHTCGGILLNSHWVLTAAHCFKNKKKVTDWRLIFGANEVVWGSNKPVKPPLQERFVEEIIIHEKYVSGLEINDIALIKITPPVPCGPFIGPGCLPQFKAGPPRAPQTCWVTGWGYLKEKGPRTSPTLQEARVALIDLELCNSTRWYNGRIRSTNVCAGYPRGKIDTCQGDSGGPLMCRDRAENTFVVVGITSWGVGCARAKRPGVYTSTWPYLNWIASKIGSNALQMVQLGTPPRPSTPAPPVRPPSVQTPVRPPWYFQRPPGPSQQPGSRPRP27 residues PAPPPAPPPPPPPPPPPPPPPPPPPQQ MW 2648.4.https://www.gtexportal.org/home/gene/ACR The human acrosin gene is expressed in testis and to a small extent in mammary breast tissue, lung, spleen, adipose tissue, and tibial artery.Acrosin is the major proteinase present in the acrosome of mature spermatozoa. It is a typical serine proteinase with trypsin-like specificity. It is stored in the acrosome in its precursor form, proacrosin. The active enzyme functions in the lysis of the zona pellucida, thus facilitating penetration of the sperm through the innermost glycoprotein layers of the ovum. The mRNA for proacrosin is synthesized only in the postmeiotic stages of spermatogenesis. In humans proacrosin first appears in the haploid spermatids. Image 2SPRAPAPPPSGALLPEPGQRCEAVSSSPPPPPCAQNPLHPSPSHSACKEPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDHKLPNTKIRSGGPASAAGGPPGRPNGGPPAL.>XP_003131596.1 PREDICTED: homeobox protein Hox-B4 [Sus scrofa] MAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESSFQPEAGFGRRAACTVQRYAAC21 residues RDPGPPPPPPPPPPPPPPPGL MW 2069.1.The HOXB4 gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on human chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. Intracellular or ectopic expression of this protein expands hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion.Image 3SPPFQLTKPGLWSTLHGDAWGPERKGTAPPERQEQRHSLPRHPYPYPAPAYPSRTPWPRLVPAAPPGPGPXPPGAESHGCPPATRPPGSDLRESRVQRSRMDSSVSPAATTACVPYAPSRPPALPGTTTSSSSSSSNTGLRGVEPSPGIPGADHYQTPALEVSSHQGRLGPSAHSSRKPFLAAPAATPHLSLPPGPPSPPPPPCPRLLRPPPPPAWLKGPACRAAREDGEILEELFFGAEGRPRPPPPPLPHREGFLGPPAPRFSVGTQDSHTPPTPPTTSSSSSNNGSHSSSPTGSVSFPPPPYLARSMDPLPRPPSPTLSPQDPPLAPLSLALPPAPPSSCHQNTSGSFRRPESPRPRVSFPKTPEVGPGPSPGPLNKAPQPVPSRVGELPARGPRLFDFPPTPLEDQFEEPAEFKILPDGLANIMKMLDESIRKEEEQQQQEAGVVPPPPLKEPFASLQPPFPTDTAPATTAATTAATTTATQEEEKKPPPALPPPPPLAKFPPPPQPQPPPPPLPPPASPASLLKSLASVLEGQKYCYRGTGAAVATRPGPLPTTQYSPGPPSGATAPPPTSAAPSAQGSPQPSASSSSQFSTSGGPWARERRAGEEPAPGPTTPAPPPPPLPLPPARSESEVLEEISRACETLVERVGRGATDPADPADTADPVDTGAERLLPPAQAKEEAGGASAVAAAAAGPGSSKRRQKEHQKEHRRHRRACKDSVGRRPREGRAKAKAKAPKEKSRRVLGNLDLQSEEIQGREKARPDLGGASKAKPPTAPAPLPAPAPSTQSTPPSAPVPGKKAREEAPGPPGVSRADMLKLRSLSEGPPKELKIRLIKVESGDKETFIASEVEERRLRMADLTISHCAADVVRASKNAKVKGKFRESYLSPAQSVKPKINTEEKLPREKLNPPTPSIYLESKRDAFSPVLLQFCTDPRNPITVIRGLAGSLRLNLGLFSTKTLVEASGEHTVEVRTQVQQPSDENWDLTGTRQIWPCESSRSHTTIAKYAQYQASSFQESLQEEKESEDEESEEPDSTTETPPSSAPDPKNHHIIKFGTNIDLSDAKRWKPQLQELLKLPAFMRVTSTGNMLSHVGHTILGMNTVQLYMKVPGSRTPGHQENNNFCSVNINIGPGDCEWFAVHEHYWETISAFCDRHGVDYLTGSWWPILDDLYASNIPVYRFVQRPGDLVWINAGTVHWVQATGWCNNIAWNVGPLTAYQYQLALERYEWNEVKNVKSIVPMIHVSWNVARTVKISDPDLFKMIKFCLLQSMKHCQVQRESLVRAGKKIAYQGRVKDEPAYYCNECDVEVFNILFVTSENGSRNTYLVHCEACARRRSAGLQGVVVLEQYRTEELAQAYDAFTLAPASTSR.>XP_005657086.1 PREDICTED: LOW QUALITY PROTEIN: lysine-specific demethylase 6B [Sus scrofa] MHRAVDPPGARTAREAFALGGLSCAGAWSSCPPHPPPRSAWLPGGRCSASIGQPPLSAPLPPSHGSSSGHPNKLYFAPGTPNPRPLHGKLESLHGCVQALLREPAQPGLWEQLGQLYESEHDSEEAIRCYHSALRYGGSLAELGPRIGRLQQAQLWNFHAGSCQHRPKVLPPLEQVWNLLHLEHKRNYGAKRGGPPVKRAAEPPVVQPVPPAALSGPSGEEGLSPGGKRRRGCNSEQTGLPPGL28 residues PLPPPPLPPPPPPPPPPPPPPPLPGLAT MW 2737.5.Histone demethylase specifically demethylates ׳Lys-27׳ of histone H3, thereby playing a central role in histone code . Demethylates trimethylated and dimethylated H3 ׳Lys-27׳ . Plays a central role in regulation of posterior development, by regulating HOX gene expression (PubMed:17851529). Involved in inflammatory response by participating in macrophage differentiation in case of inflammation by regulating gene expression and macrophage differentiation (PubMed:17825402). Plays a demethylase-independent role in chromatin remodeling to regulate T-box family member-dependent gene expression by acting as a link between T-box factors and the SMARCA4-containing SWI/SNF remodeling complex.Image 4PPRVQLPVSLDLPLFPSIMMQPVQHPALPPQLALQLPQMDTLSADLTQLCQQQLGLDPNFLRHSQFKRPRTRITDDQLKILRAYFDINNSPSEEQIQEMAEKSGLSQKVIKHWFRNTLFKERQRNKDSPYNFSNPPITVLEDIRIDPQPSSLEHYKSDASFSKRSSRTRFTDYQLRVLQDFFDTNAYPKDDEIEQLSTVLNLPTRVIVVWFQNARQKARKSYENQAEAKDNEKRELTNERYIRTSNMQYQCKKCNVVFPRIFDLITHQKKQCYKDEDDDAQDESQTEDSMDATDQVVYKHCTVSGQTEAAKNAPVAAASSGSGASTPLLPSPKPEPEKTSPKPEYPTEKPKQSDPSPPSQGTKPALPLASTSSEPPQAAAAQPQPQPPKQPQLIGRPPSASQTPIPSSPLQISMTSLQNSLPPQLLQYQCDQCTVAFPTLELWQEHQHMHFLAAQNQFLHSPFLERPMDMPYMIFDPNNPLMTGQLLSGFLTQMPPQNASSQTPASATVAASLKGNWDDKEDTNCSEKEGGNSGEDQHRDKRXRTTITPDKLEILYEKYLLDSNPTRKMLDHIAREVGLKKRVVQVWFQNTRARERKGQFRAVGPAQSHKRCPFCRALFKAKSALESHIRSRHWNEGKQAGYSLPPSPLIATEDGGESPQKYIYFDYPSLPLTKIDLSSENELASTVSTPVSKTAELSPKNLLSPSSFKAECSEDVENLNAPPAEAGYDQNKPDFDETSSINTAISDATTGDEGNAEMESTTGSSGDVKPALSPKEPKTLDTLAKTATTPTTEVCDEKFLFSLTSPSIPFNDKDGDHDQSFYITDDPDDNADRSETSSIADPSSPNPFGSSNPFKSKSNDRPGHKRFRTQMSNLQLKVLKACFSDYRTPTMQECEMLGNEIGLPKRVVQVWFQNARAKEKKFKINIGKPFMINQSGTEGTKPECTLCGVKYSARLSIRDHIFSKQHISKVRETVGSQLDREKDYLAPTTVRQLMAQQELDRIKKASDVLGLAVQQPSMMDSSSLHGISLPAAYPGLPGLPPVLLPGMNGPSSLPGFPQNSNTLTPPGAGMLGFPTSATSSPALSLSSAPTKPLLQImage 5SGQQTEPQNKESEKKQTKPNKVKKIKEEELEATKPEKHPKKEEKISSALSVLGKVVGETHVDPSQLQALQNAIAGDPASFLGGQFLPYFIPGFASYFTPQLPGTVQGGYLPPVCGMESLFPYGPTMPQTLAGLSPGALLQQYQQYQQNLQDSLQKQQKQQQQEQPQKPGQAKTSKGESEPPQNASDASETKEDKSTATESTKEEPQLESKSADFSDTYVVPFVKYEFICRKCQMMFTDEDAAVNHQKSFCYFGQPLIDPQETVLRVPVSRYQCLACDVAISGNEALSQHLQSSLHKEKTIKQAMRNAKEHVRLLPHSVCSPNPNTTSTSQSAASSNTYPHLSCFSMKSWPNILFQASARRAASSPSSPPSLSLPSTVTSSLCSTSGVQTSLPTESCSDESDSELSQKLEDLDNSLEVKAKPASGLDGNFNSIRMDMFSV.>XP_005663076.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger homeobox protein 4 [Sus scrofa] METCDSPPISRQENGQSTSKLCGTAQLDNEVPEKVAGMEPDRENSSTDDNLKTDERKSEVLLGFSVENAAATQVTSAKEIPCNECATSFPSLQKYMEHHCPNARLPVLKDDNESEISELEDSDVENLTGEIVYQPDGSAYIIEDSKESGQNAQTGANSKLFSTAMFLDSLASAGEKSDQSASAPMSFYPQIINTFHIASSLGKPFTADQAFPNTSALAGVGPVLHSFRVYDLRHKREKDYLTSDGSAKNSCVSKDVPNNVDLSKFDGCVSDGKRKPVLMCFLCKLSFGYIRSFVTHAVHDHRMTLNEEEQKLLSNKCVSAIIQGIGKDKEPLISFLEPKKSTSVYPHFSTTNLIGPDPTFRGLWSAFHVENGDSLPAGFAFLKGSAGTSGSAEQPLGITQMPKAEVTLGGLSSLVVNTPITSVSLSNASSESSKMSESKDQENDCERPKESNALHPNGECPVKSEPTEAGDEDEEDAYSNELEDEEVLGELTDSIGNKDFPLLNQSISPLSSSVLKFIEKGPSSSSASVTDDAEKKKPTAAVRASGGVANSYGIGGKDFAEASASKDGATAAHSSEPARGDEDSSATPHQHGFTPSAPGTPGPGGDGSPGSGIECPKCDTVLGSSRSLGGHMTMMHSRNSCKTLKCPKCNWHYKYQQTLEAHMKEKHPEPGGSCVYCKTGQPHPRLARGESYTCGYKPFRCEVCNYSTTTKGNLSIHMQSDKHLNNVQNLQNGNGEQVFGHSAPAPNTSLSGCGTPSPSKPKQKPTWRCEVCDYETNVARNLRIHMTSEKHMHNMMLLQQNMKQIQHNLHLGLAPAEAELYQYYLAQNIGLTGMKLENPGDPQLMLNPFQLDPATAAALAPGLVNNELPPEIRLASGQLMGDDLSLLTAGELSPYISDPALKLFQCAVCNKFTSDSLEALSVHVSSERSLPEEEWRAVIGDIYQCKLCNYNTQLKANFQLHCKTDKHMQKYQLVAHIKEGGKSNEWRLKCIAIGNPVHLKCNACDYYTNSVDKLRLHTTNHRHEAALKLYKHLQKQEGAVNPESCYYYCAVCDYSTKVKLNLVQHVRSVKHQQTEGLRKLQLHQQGLAPEEDNLSEIFFVKDCPPNELETASLGARTCEDDLLEQQLRAPSEEQSEETEGASRPTAVAEDDEKDTSERDNNEGKNSNKDTGIITPEKELKVSVAGGTQPLLLAKEEDVATKRSKPTEDSKFCHEQFYQCPYCNYNSRDQSRIQMHVLSQHSVQPVICCPLCQDVLSNKMHLQLHLTHLHSVSPDCVEKLLMTVPVPDVMMPNSLLLPAAASEKSERDTPAAITAEGPGKYSGESPMDDKSMAGLDDSKAIMEIKSEEQKPTKEPTEASEWNKNSSKDGKISDPLQDQLSEQQKRQPLSVSDRHVYKYRCNHCSLAFKTMQKLQIHSQYHAIRAATMCNLCQRSFRTFQALKKHLEAGHPELSEAELQQLYASLPVNGELWAESETMAQDDHALEQEMEREYEVDHEGKASPVGSDSSSIPDDMGSEPKRTLPFRKGPNFTMEKFLDPSRPYKCTVCKESFTQKNILLVHYNSVSHLHKLKKVLQEASSPVPQETNSSTDNKPYKCSICNVAYSQSSTLEIHMRSVLHQTKARAAKLEPSSHVVSGHSAANVSSPGQGMLDSMSLAGVSSKDTHLDAKELNKKQTPELISAQPAHHPPQSPAQIQMQLQHELQQQAAFFQPQFLNPAFLPHFPMTPEALLQFQQPQFLFPFYIPGTEFSLGPDLGLPGSAAFGMPGMTGMAGSLLEDLKQQIQTQHHVGQTQLQILQQQAQQYQATQPQLQSQKPQQQPQPQPQQQQASKLLKQEQTTLASAECPIVKDIPSFKEAEEMAKKQDKPKQEVXSEGEGLKEGKDEKKQKSSEPSILPPRIASGARGNAAKALLENFGFELVIQYNENRQKVQKKGKSGEGESTEKLECGTCGKLFSNVLILKSHQEHVHGQFFPYGALEKFARQYREAYDKLYPISPSSPETPPPPPPPPPLPPAPPQPASLGPVKLPSTVSTPIQAPPP22 residues TPPPPPPPPPPPPPPPPPPPSA MW 2121.1.18 residues TPPPPPPPPPPPPPPSSL MW 1764.9.May play a role in neural and muscle differentiation. May be involved in transcriptional regulation.Image 6PMPVHEPLSPQQLQQQQDMYNKKIPSLFEIVVRPTGQLAEKLGVRFPGPGGPPGPMGPGPNMGPPGPMGGPMHPDMHPDMHPDMHPDMHPDMPMGPGMNPGPPMGPGGPPMMPYGPGDSPHSGMMPPIPPAQNFYENFYQQQEGMEMEPGLIGDTEDYGHYEELPGEPGEHLFPEHPLEPDSFSEGGPPGRPKPGAGVPDFLPSAQRALYLRIQQKQQEEEERARRLAESSKQDRENEEGDPGNWYSSDEDEGGSSVTSILKTLRQQTSSRPQASGGELSSSGLGDPRLQKGHPTGGRLADPRLSRDPRLSRHAEASGGSGPGDTGPSDPRLARSLPTPRPKGGLHSSPGGPSGSKGSGPPPAEEEEGERALREKAVNIPLDPLPGHPLRDPRSQLQQFSHIKKDVTLSKPSFARTVLWNPEDLIPLPIPKQDAVPPVPVALQSMPALDPRLHRTTTTSGPPNPRQRPGTSTDPSASGSNLPDFELLSRILKTVNATGPSAAPGPGDKPSDPRVRKTPTDPRLQKPADSATSSRAAKPGSTEVSPSASPSGESSPPATAPYDPRVLAAGGLGQGSGSGQSSVLSGISLYDPRTPNAGGKATEPAADTGTQPKGPEGNGKSAATKAKEPPFVRKSALEQPESGKPGADGGAAAATDRYNSYNRPRPKATPAAAASGTPPPEGASPQPGVHNLPVPTLFGTVKQAPKTGSGSPFAGNSPAREGEQDAGSLKDVFKGFDPTASPFCQ.>XP_005664683.1 PREDICTED: LOW QUALITY PROTEIN: zinc finger CCCH domain-containing protein 4 [Sus scrofa] MEAAPGTPPPPPSESPPPPSPPLPSTPSPPPCSPDACPATPHLLHHRLPLPDDREDGELEEGELEDDGAEETQDTSGGPERSRKEKGDKHHSDSDEEKSHRRLKRKRKKEREKEKRRSKKRRKSKHKRHASSSDDFSDFSDDSDFSPSEKGHRKYREYSPPYAPSHQQYPPSHTTPLPKKAYSKMDSKGYSMYEDYENEQYGEYEGDEEEDMGKDDYDDFTKELNQYRRAKEGSSRGRGSRGRGRGYRGRGSRGGSRGRGMGRGSRGRGRGSMGGDHPEDDEDFYEEEMEYGESEEPMGDEDYDDYSKELNQYRRSKDGRGRGLSRGRGRGSRGRGKGMGRGRGRGGSRGGMNKGGMNEDDDFYDEDMGDGGGGGGSYRRSDHDKPHQQSDKKGKVICKYFVEGRCTWGDHCNFSHDIELPKKRELCKFYITGFCARAENCPYMHGDFPCKLYHTTGNCINGDDCMFSHDPLTEETRELLDKMLADDAEAGAEDEKEVEELKKQGINPLPKPPPGVGLLPTPPRPPGPPAPTSPNGRPLQ19 residues GGPPPPPPPPPPPPGPPQM MW 1806.9.NCBI and UniProt have ZC3H4 zinc finger CCCH-type containing protein 4 (Homo sapiens).This gene encodes a member of a family of CCCH (C-x8-C-x5-C-x3-H type) zinc finger domain-containing proteins. These zinc finger domains, which coordinate zinc finger binding and are characterized by three cysteine residues and one histidine residue, are nucleic acid-binding. Other family members are known to function in post-transcriptional regulation.Image 7GRTPPTLLSTLQYPRPSSGTLASASPDWAGPGARLRQQSSSSKGDSPELKPRAVHKQGPSPVSPNALDRTAAWLLTMNAQLLEDEALGPDPPHRDRLRSKEELSQAEKDLAVLQDKLRISTKKLEEYETLFKCQEETTQKLVLEYQARLEEGEERLRRQQEDKDIQMKGIISRLMSVEEELKKDHAEMQAAVDSKQKIIDAQEKRIASLDAANARLMSALTQLKERYSMQARNGISPTNPTKLQITENGEFRNSSNC.>XP_003353684.3 PREDICTED: LOW QUALITY PROTEIN: disabled homolog 2-interacting protein-like isoform 1 [Sus scrofa] MSAGGSARKSTGRPSYYYRLLRRPRLQRQRSRSRSRTRPARESPQERPGSRRSLPGSLSEKSPSMEPSAATPFRVTGFLSRRLKGSIKRTKSQPKLDRNHSFRHILPGFRSAAAAAAAASAADNERSHLMPRLKESRSHESLLSPSSAVEALALSMEEEVVIKPVHSSILGQDYCFEVTTSSGSKCFSCRSAAERDKWMENLRRAVHPNKDNSRRVEHILKLWVIEAKDLPAKKKYLCELCLDDVLYARTTGKLKTDNVFWGEHFEFHNLPPLRTVTVHLYRETDKKKKKERNSYLGLVSLPAASVAGRQFVEKWYPVVTPNPKGGKGPGPMIRIKARYQTITILPMEMYKEFAEHITNHYLGLCAALEPILSAKTKEEMASALVHILQSTGKVKDFLTDLMMSEVDRCGENEHLIFRENTLATKAIEEYLKLVGQKYLQDALGEFIKALYESDENCEVDPSKCSAADLPEHQGNLKMCCELAFCKIINSYCVFPRELKEVFASWRQECSSRGRPDISERLISASLFLRFLCPAIMSPSLFHLLQEYPDDRTARTLTLIAKVTQNLANFAKFGSKEEYMSFMNQFLEHEWTNMQRFLLEISNPETISNTAGFEGYIDLGRELSSLHSLLWEAVSQLEQSIVSKLGPLPRILRDVHTALSTPGSGQLTGTNDLASTPGSGSSSISAGLQKMVIENDLSGLIDFTRLPSPTPENKDLFFVTRSSGVQPSPARSSSYSEANEPDLQMANGGKSLSMVDLQDARALDGEAGSPAGPDALAADGQVPTAQLVAGWPARAAPVSLAGLATVRRAGQTPTTPGTSEGAPGRPQLLAPLSFQNPVYQMAAGLPLSPRGLGDSGSEGHSSLSSHSNSEELAAAAKLGSFSS;ATAAAAEDLGRRPGELARRQMSLTEKGGQPTVPRQNSAGPQRR16 residues IDQPPPPPPPPPPAPR MW 1668.9.Functions as a scaffold protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways.Image 8LALSEDTEPSSSESRTGLCSPEDNSLTPLLDEVAAPEGRAATVPRGRGRSRGDSSRSSASELRRDSLTSPEDELGAEVGDEAGDKKSPWQRREERPLMVFNVK.>XP_005668832.1 PREDICTED: protein FAM171A2 [Sus scrofa] MPPPSGPSVLARLLPLLGLLLGGASRAPGKSPPEPPSPQEILIKVQVYVSGELVPLARASVDVFGNRMLLAAGTTDSEGVATLPLSYRLGTWVLVTAARPGFLTNSVPWRVDKLPLYASVSLYLLPERPATLILYEDLVHILLGSPGARSQPWVQFQRRAARLPVSSTYSQLWASLTPASTQQEMRAFPAFLGTDASSSGNGSWLELMPVAAVSVHLLAGNGTEVPLSGPIHLSLPVPSEPRALAVGTSIPAWRFDPKSGLWVRNGTGVIRKEGRQLYWTFISPQLGYWAAAMASPTSGLVTITSGIQDIGTYHTIFLLTILAALALLVLILLCLLIYYCRRRCLKPRQQHRKLQLSGPSDGNKRDQATSMSQLHLICGGPLEPAASGDPEAPPPGPLHSAFSSSRDLAASRDDFFRAKPRSASRPAAEPAGARGGEGAGLKGARSVEGPGGLEPGLEEYRRGPPGTATFLQEPPSPPPPFEHYLGHKGAAESKTPDFLLSQSVDQLERPPSLSQAGQLIFCGSIDHHSQVRHSYIDLQAGGGGRSTDASLDSGVDVHEARPARRRPLREERERAA17 residues AAAPPPPPPPPPPAPPR MW 1622.9.Image 9CPYSGAGFPPAPPPPPPPPLPGGPPVPPPPPGLPPPSHLNGYSHLGKKKRMRSFFWKTIPEEQVRGKTNIWTLAARQQHHYQIDTKTIEELFGQQEESAKSSPSRRGGPLNSSFREAREEITILDAKRSMNIGIFLKQFKKSPPSIVEDIHQGKSEHYGSETLREFLKLLPESEEIKKLKAFSGDVAKLSLADSFLHCLIQVPNYSLRIEAMVLKKEFLPSCSSLYTDMTILRTATKELMSCEELHSILHLVLQAGNIMNAGGYAGNAVGFKLSSLLKLADTKANKPGMNLLHFVAQEAQKKDAVLLNFSEKLHHVQEAARLSLDNTEAELHSLFVRTRSLKENIQRDGELCQQMEDFLQFAVEELSELERWKQELLAEAHTLIDFFCEDKDTVKLDECLQIFRDFCIKFNKAVKDNHDREVQELKQLQRLKEQEQKRRSWAAGELGFSRSSSENDVELLTKRGAEDPFLHSRPISPSHRPPNTRRSRLSLGASADRELLTFLESSTGNPEELKFNSLPRSCPRQAPPSRAWMESGEQRDQDSSQAHRLPASKDQEEATDPPSTWQSQLLAPRLEEPATALPRVRRSGVSILRKRNSEPLGLGPVRSPPLSPLALGIKEHELVTGLAQFDLQAPKGPEEPARLTMNDFSPMELMSVVGESPQAPRAPNDHRCEGLIPPCFSNEDLGNILLYVRAHAASRPYRESRAPSRSSFRKPSVKPLRNVPRPKPDEDKMCRSSSQGPESPEEAPRAPAAPSAPRGPAPVPSFARNTVASSSRCLRTDSPAVARPPGLTRTVSQRQLRAKGGPEEAAPKDGGALRRASSARGPRKGPELPEGPRAGSEASPKGRGAGERASVRLKDASRPALGKGLHPLRK.>XP_005666867.1 PREDICTED: FH2 domain-containing protein 1 [Sus scrofa] MHVMNCVSSVSDKGNGNIAPAAGFMIGQT14 residues PPPPSPPPPPPPPP MW 1366.7.Formin-homology-domain-containing protein FHOD1 is involved in cell migration and adhesion, acting as a regulator of stress fibers organization, maturation of integrin-based adhesion sites, and podosome-associated contractility.Image 10GGLTSPIFCSTKPKKLLKTSSFHLLRRRDPPFQTPKKLYAQEYEFEADEDKADVPADIRLNPRRLPDLVSSCRSRPALSPLGDIDFCPPNPGPDGPRRRGRKPTKAKRDGPPRPRGRPRIRPLEGPATAGPALASTPTDGAKKPRGRGRGRGRKAEEAGGTRLEPLKPLKIKLSVPKAGEGLGASSGEAVSGADPNSLDSSLTREKIEAKIKEVEEKQPEMKSGFMASFLDFLKSGKRHPPLYQAGLTPPLSPPKSVPPSVPARGLQPQPPSTPAVPHPPPAGAFGLGGALEAAESEGLGLGCPSPCKRLDEELKRNLETLPSFSSDEEDSVAKNRDLQESISSAISALDDPPLAGPKDTSTPDGPPLAADAAVPGPPPLPGLPSASSNGTPEPPLLEEKPPPSPPPVPTPQPPPPPPALPSPPPLVAPAPSSPPPQImage 11PPAPAPAPAPPALPSPPAPPPPPAAAAAPPPEEPAAPSPDDSEPPDARPLHLAKKQETAAVCGETDEEAGESGGEGIFRERDEFVIRAEDIPSLKLALQTGREPPPIWRVQKALLQKFTPEIKDGQRQFCATSNYLGYFGDAKNRYQRLYVKFLENVNKKDYVRVCARKPWHRPPVPVRRSGQAKGPSSSGGSSAPPPKAPAPPPKPETPDKMASEKPPEQTPETAVPEPPAPEKPSPPRLVEKEKEKERTPRGERPLRGERGTGGRQIRPDRGLTTGQPATSRLPKSRPTKVKAEPPPKKRKKWLKEAAGNASAGGGPPGSSSDSESSPGAPSEDERAVPGRLLKTRAMREMYRSYVEMLVSTALDPDMIQALEDTHDELYLPPMRKIDGLLNEHKKKVLKRLSLSPALQDALHTFPQLQVEQSGEGSPEEGAVRLRPAGEPYNRKTLSKLKRSVVRAQEFKVELDKSGYYTLYHSLHHYKYHTFLRCRDQTLAIEGGAEDLGQEEVVQQCMRNQPWLEQLFDSFSDLLAQAQAHSRCG.>XP_003127395.2 PREDICTED: proline-rich protein 12 isoform X1[Sus scrofa] MDRNYPSAGFGDPLGAGAGWSYERSAKASLVYGSSRTSHPETDILHRQAYAAPHPLQSYATNHHPAGLSGLFDTGLHHAGSAGPDASVMNLISALESRGPQPGPSASSLLSQFRSPSWQTAMHTPGPTELFISGALPGSSTFPSSSALSAYQHPASFGSRPFPVPSSLSLQDPPFSPPANGLLSPHDVLHLKPSQAPTVPSSLGFERLAGGGVLGPAGLGPAQTPPYRPGPPDPPPPPRHLPTQFNLLASSSAAAAAAAEQSSPQLYNFSGAAPGPPPPERALPRQDTVIKHYQRPASAQPPPPPPPAHALQHYLSCGGSYPSMGHRANLACSPLGGGEPSPGAGEPSKAGPSGATAGASGRAAGPEAAGGGGAGGGGGGYRPIIQSPGYKTGKGGYGAAAGGANRPPPPRSTATPKCQSLGGPAAAYATGKASGAGGAGGQAYSPGQPQGLLGPQAYGQGFGGGQAQDLSKGPSYSGGPQQPPNGPPPPGLATCQSYSPDQLQGQLYGVQGEPYPGPAAHSQGLPTASPSLSYSTGHSPALSGHGGGWGPSSLGGGGEASPSHIIRPLQSPPAPGRPPGVGSPGAPGKYLSSVLASAPFLAPPGAGSYAAGAGGYKGKGDGSELLAGPGGPPAERTEDEEFLIQHLLQAPSPPRTSGADGLVGEDGAADASKGLGGSGGAGGPPGTPYELAKEDPQRYHLQSVIRTSASLDEGATAALELGLGRLKEKKKGPERGGETPEGLATSVVHYGAGAKELGAFLQKSPPPPPPTAQSAQPTPHGLLLEAGGPDLPLVLPPPPPQLLPSVLSHAPSPSSSAPKVGVHLLEPAARDGAPPPPPPPPPPPMPLQLEAHLRSHGLEPGAPSPRLRPEESLEPPGAMQELLGALEPLPPGPGDTGVGPPTAEGKDPSGAYRSPSPQGTKAPRFVPLTSICFPDSLLQDEERSFFPTMEEMFGGGPADDYGKAGPPEDEGDPKAGAGPPPGPPAYDPYGPYCSSRASGAGPETPGLGLDPSKPPELPSTVNAEPLGLIQSGPHQA19 residues APPPPPPPPPPPPPASEPK MW 1863.0.20 residues LPPPPPPPPPPPPPPPPPPP MW 1975.1.Image 12NQANTELPKLSRDEQRGRGALLQDICKGTKLKKVTNINDRSAPILEKPKGSSGGYGPGAAALQPKGGLFQGGVPKLRPVGAKDGSENLAGKPALQVPSSRAAAPRPPVSTASGRPQDDTDSNRASLPELPRTQRPSLPDLSRPHATSSTGMKHSSSAPPPPPPGRRANAPPTPLAMHSNKAPAYNREKPLPPTPGQRLHPGREGPSAPPPVKPPPSPVNIRTGPSGQSLAPPPPPYRQPPGVPNGPSSPTNESAPELPQRHNSLHRKTPGPVRGLAPPPPTSASPSLQSNRPPPPARDPPSRGAAPPPPPPMIRNGARDAPPPPPPYRMHGSEPLSRGKPPPPPSRTPAGPPPPPPPPLRNGHRDSITTVRSFLDDFESKYSFHPVEDFPAPEEYKHFQRVYPSKTNRAARGAPPLPPILR.>NP_001231241.1 WAS/WASL-interacting protein family member2 Sus scrofa 19 residues MPIPPPPPPPPGPPPPPTF MW 1926.0.Plays an active role in the formation of cell surface protrusions downstream of activated PDGFB receptors. Plays an important role in actin-microspike formation through cooperation with WASL. May cooperate with WASP and WASL to induce mobilization and reorganization of the actin filament system.Image 13LTPVKCEDPQRVVPTVNPVKTNGTLLRNGGFPGAPNKIPNGDICCKPGSIVDKAPVQPLMHRPEKDRCPQAGPRERVRFNEKVQYHGYCPDCDTRYNIKNREVHLHSEPVHPPGKLPPQGPHHPPPPHLPPFPLENGGLGISHSNSFPPLRPATVPPPTAPKPQKTILRKSTTTTV.>XP_005655053.1 PREDICTED: proline-rich protein 16 [Sus scrofa] MTDSSKTDTLNSSSSGTTASSIEKIKVQANAPLIKPPAHPSAILTVLRK9 residue PNPPPPPPR MW 967.5.Regulator of cell size that promotes cell size increase independently of mTOR and Hippo signaling pathways. Acts by stimulating the translation of specific mRNAs, including those encoding proteins affecting mitochondrial functions. Increases mitochondrial mass and respiration.Image 14LLSAPAPNATSCPAEESWWSGLAIVIAVCCASLVFLTVLVIICYKAIKRKPLRKEENGTSVAEYPMTSSQSNKGVDVNSAVV.>XP_020955308.1 proline-rich membrane anchor 1 isoform X2 [Sus scrofa] MLLRDLVLRRGCCWPSLLLHCALHPLWGFVQVAHGEPQKSCSKVTDSCQHICQCR16 residues PPPPLPPPPPPPPPPR MW 1645.9.Required to anchor acetylcholinesterase (ACHE) to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses in brain. Also able to organize ACHE into tetramers.


Dumbbell-shaped DNA minimal vectors represent genetic vectors solely composed of the gene expression cassette of interest and terminal closing loop structures. Dumbbell vectors for small hairpin RNA or microRNA expression are extremely small-sized, which is advantageous with regard to cellular delivery and nuclear diffusion. Conventional strategies for the generation of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell production. Here, we present a novel cloning-free method for the generation of small RNA-expressing dumbbell vectors that also does not require any restriction endonucleases. This new PCR-based method uses a universal DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are introduced by the PCR primers. Dumbbells are formed by denaturing and reannealing of the PCR product and are covalently closed using ssDNA ligase. The new protocol generates plus- and/or minus-strand dumbbells, both of which were shown to trigger efficient target gene knockdown. This method enables fast, cheap production of small RNA-expressing dumbbell vectors in a high throughput-compatible manner for functional genomics screens or, as dumbbells are not prone to transgene silencing, for knockdown studies in primary cells.  For shRNA gene delivery, researchers explore viral or non-viral delivery vectors. While viral vectors are costly and often trigger immune responses or pose the risk of genomic vector integration, many non-viral delivery vectors involve non-nucleic acid helper functions that can be toxic to the cells.Here, we report a cloning-free method for the generation of shRNA-expressing dumbbell vectors. This PCR-based method uses a universal template and sequences coding for a specific shRNA are introduced by the PCR primers. This novel protocol produces size-minimized hairpin-template transcribing dumbbells, does not require any restriction or nicking endonucleases, and is high throughput compatible.5), and (3) the hsa-miR-30 precursor stem  in which the self-complementary sequence portions were separated from each other A–S1C. Paequenced D and S1ENext, we aimed to PCR-amplify the universal template using primers that introduced the sequence coding for a published firefly luciferase-targeting shRNA.With the decision to use either a 5′-phosphorylated forward primer, a 5′-phosphorylated reverse primer, or two phosphorylated primers for PCR, only (1) plus-strand-derived dumbbells, (2) minus-strand-derived dumbbells, or (3) a mix of both can be generated  and S2. Employing the above protocol using either phosphorylated forward or reverse primers, we generated both plus- and minus-strand-derived luciferase- or lamin A/C-targeting dumbbells in separate reactions  and 4. TThe protocol described here combines all the advantages of previously reported protocols for dumbbell vector production. It represents (1) a cloning-free protocol that (2) does not involve any restriction or nicking endonucleases, (3) employs an efficient intra-molecular ligation reaction, and (4) allows production of extremely small hairpin template-transcribing dumbbell vectors. The previously described gap-primer PCR protocol also involves an intra-molecular ligation but requires a cloning step for the generation of every new vector, and it is not suitable to generate hairpin template-transcribing vectors due to the presence of abasic sequence positions.We demonstrate the proof-of-principle that this new method can generate partly mismatched shRNA-expressing dumbbell vectors, indicating the technology might also be explored for the generation of miRNA-expressing dumbbells. Mismatches in dumbbell vectors were reported earlier and demonstrated not to impair vector activity.We observed that among the luciferase- or lamin A/C-targeting dumbbells, the minus- or plus-strand-derived dumbbell exhibited a stronger target gene knockdown activity, respectively. This difference might be assigned to differences with regard to the efficiency and accuracy of endogenous shRNA processing by Dicer, which depends on the sequence and structure of shRNA loops and stems. Here, we employed the hsa-miR-30 stem, as miRNA stems were reported to facilitate shRNA processing and knockdown activities in most of the cases.In conclusion, this novel method efficiently generates size-minimized hairpin template-transcribing dumbbells in a short period of time and at low costs and can be explored for the parallelized production of shRNA or miRNA expression vectors for functional genomics screens or drug development.AGCTTCGCGCTCACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGTT-3′; oligo UT2, 5′P-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3′; oligo UT3, 5′P-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3′; oligo UT4, 5′-GATCCTTTGCCTACTGAGAAGATTTTTCTGTGCTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTGATTTCCCAGAACACATAGCGACATGCAAATATGAATTGTCCAGAAAACT-3′. Bold indicates HindIII and BamHI compatible overhangs; underlined indicates dumbbell loop-forming tetranucleotide. The 5′ phosphorylated oligos UT2 and UT3 were hybridized with the complementary oligos UT1 and UT4, respectively. The resulting UT1/UT2 and UT3/UT4 duplexes were ligated to form the universal template sequence bearing HindIII and BamHI-compatible 5′ overhangs, which was subsequently cloned into pVax1 , yielding the universal template vector pVax1-UT  endonucleolytic cleavage followed by analytical agarose gel electrophoresis, which yielded the expected insert size of 262 bp, and by sequencing of the ligation sites  A and S1BpVax1-UT B. For clon sites C and S1DtgaaggctcctcagaaacagctcCGCGCTCACTGAGAAGATTT-3′; FP_Lamin 5′-tgaaagcccagatcgtcaccacccgcCGCGCTCACTGAGAAGATTT-3′; reverse primers, RP_Luciferase 5′-agagaggctcctcagaaacagctcTTTGCCTACTGAGAAGATTTTTCTGT-3′. RP_Lamin 5′-agagaagcccagatcgtcaccaccttTTTGCCTACTGAGAAGATTTTTCTGT-3′.Luciferase- or lamin A/C-specific primers were synthesized by AITbiotech (Singapore) or IDT (Singapore). Uppercase letters indicate the universal template binding sites, and lowercase letters indicate the shRNA coding sequences in which the loop-forming nucleotides are underlined: forward primers, FP_Luciferase 5′-Two blocking ODNs  were added to the PCR to suppress refolding and self-priming of the universal template strands: Block_1, 5′-GGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTGAGCGCGA-3′; Block_2, 5′-TTCTGGACAATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGAGCACAGAAAAATCTTCTCAGTAGGCAAAG-3′.Primers for the quantification of luciferase and β-actin mRNA levels were synthesized by AITbiotech (Singapore). PCR forward primers are as follow: qPCR_FP_Luciferase 5′-CGCTGGGCGTTAATCAAAGA-3′; qPCR_RPb-actin 5′-CTGGCACCCAGCACAATG-3′. Reverse transcription and PCR reverse primers are as follows: qPCR_RP_Luciferase 5′-GTGTTCGTCTTCGTCCCAGT-3′; qPCR_RPb-actin 5′- GCCGATCCACACGGAGTACT-3′.For the generation of strand-specific dumbbell vectors, either the forward or the reverse primers were 5′-phosphorylated. Each 50 pmol primer was incubated with 10 U T4 polynucleotide kinase  in the presence of 1 mM ATP at 37°C for 20 min followed by heat inactivation of the enzyme at 75°C for 10 min.Taq DNA polymerase (Invitrogen), 1.0 μM of each primer and blocking ODNs, 0.2 mM of each 2’-deoxyribonucleoside 5’-triphosphate , 100 ng of HindIII/BamHI cleaved pVax1-UT, 5% v/v DMSO  in a reaction volume of 30–50 μL in 1× Taq DNA polymerase buffer (Invitrogen). Linearization of pVAX1-UT usually improves the PCR yields but is not essential. Thermal cycling was carried out as follows: initial denaturation at 96°C for 5 mins 27 cycles of denaturation , annealing , and extension ; and final extension at 72°C for 10 mins. A 50-μL PCR reaction yielded about 10 μg DNA.PCR amplification of the universal template and appendage of shRNA encoding DNA was carried out using 1 U 2, and 200 mM Tris-HCl, pH 7.4), heat-denatured at 96°C for 5 min followed by gradual cooling to room temperature to allow for intramolecular folding of plus- and/or minus-strand dumbbell vectors. The resulting DNA was concentrated using ethanol precipitation, pelleted by centrifugation, and resuspended in nuclease-free water.PCR products were purified through silica-membrane-based spin columns . Purified products were diluted to 400 μL in 1× hybridization buffer , and 50 to 100 U CircLigaseII ssDNA ligase  in 1× CircLigaseII reaction buffer at 60°C for 16 h, followed by heat inactivation of the ligase at 80°C for 10 min. Highest conversion yields were observed when ligating 6 μg DNA with 100 U CircLigase.1 to 6 μg (∼10 to 60 pmol) of DNA was incubated with 2.5 mM MnClAfter ligation, products were treated with 10 U of T7 DNA polymerase  at 37°C for 1 h followed by heat inactivation at 80°C for 10 min. Products were assessed on 10% native polyacrylamide gels or 1% agarose gels, stained with ethidium bromide post-electrophoresis, and/or purified using phenol-chloroform-isoamylalcohol (25:24:1) extraction (1×), chloroform-isoamylalcohol (24:1) re-extraction (3×), and ethanol precipitation.4 cells/well were seeded in a 96-well plate. Cells were co-transfected with 100 ng of luciferase expression plasmid pGL3  and 1.5 pmol or 0.5 pmol of either plus- or minus-strand dumbbell vector DNA using Lipofectamine 2000  and a reagent:DNA ratio of 1:2.5. For the positive control (pGL3 only), empty pVAX1  was used as feeder DNA to ensure all cells received the same quantity of DNA. 48 h post-transfection, cells were washed with sterile PBS and lysed in 20 μL passive lysis buffer  for 20 min, employing gentle shaking. 10 μL of lysate was treated with 50 μL of LARII reagent , and luminescence was quantified on the Biotek reader .HEK293T cells were maintained in DMEM  supplemented with 10% (v/v) fetal bovine serum  and 1% penicillin-streptomycin antibiotic solution . 24 h prior to transfection, 2 × 10HEK293T cells were cultivated and seeded in 96-well plates 24 h prior to transfection as described above. Cells were transfected with 0.1, 0.5, or 2.5 pmol dumbbell vector DNA or 3 pmol siGENOMELamin A/C control siRNA  using Lipofectamine 3000  according to the manufacturer’s protocol. Medium was changed 24 h post-transfection, and cells were harvested after 48 h. For FACS analyses, the media was aspirated, and the cells were rinsed once with PBS before trypsinization with 50 μL of 1× trypsin-EDTA (Gibco). Trypsinized cells were collected by centrifugation at 4,200 rpm for 6 min in 200 μL media. Pelleted cells were resuspended in 100 μL media, fixed and permeabilized with intracellular fixation and permeabilization buffer set  according to manufacturer’s protocol prior to intracellular staining. To assess lamin A/C knockdown, cellular lamin A/C was stained by anti-lamin A+C antibody (ab133256) (1/200) and donkey anti-rabbit IgG H&Ls AF647 (ab150075) (1/200) . FACS was performed on LSRFortessa cell analyzer, and FACSDiva software v6.1.3  was used for the acquisition of the samples. FlowJo software V10.5.2  was used for data analyses.mfold and/or RNAfold.18Minimum free energy secondary structures of DNA and RNA were folded using the algorithms Diagrams represent mean values ± SEM of three independent experiments. The statistical analysis was performed using repeated one-way ANOVA with Tukey’s post hoc multiple comparison’s test (luciferase knockdown data) or using Student’s t test (lamin A/C knockdown data). The GraphPad Prism version 6 software  was used for the statistical analysis. p values are as indicated.V.P. developed the concept. V.P., S.L.C., A.G., P.S.L., and G.S.X.T. designed the experiments. S.L.C., A.G., P.S.L., and G.S.X.T. carried out the experiments, and all authors analyzed the data. V.P., S.L.C., A.G., and P.S.L. wrote the manuscript.The authors declare no competing interests.


Memory consolidation theory suggests that once memory formation has been completed, memory is maintained at a stable strength and is incapable of further enhancement. However, the current study reveals that even long after formation, contextual fear memory could be further enhanced. Such unexpected enhancement is possible because memory is dynamically maintained at an intermediate level that allows for bidirectional regulation. Here we find that both Rac1 activation and expression of α2-chimaerin are stimulated by single-trial contextual fear conditioning. Such sustained Rac1 activity mediates reversible forgetting, and α2-chimaerin acts as a memory molecule that reverses forgetting to sustain memory through inhibition of Rac1 activity during the maintenance stage. Therefore, the balance between activated Rac1 and expressed α2-chimaerin defines dynamic long-term memory maintenance. Our findings demonstrate that consolidated memory maintains capacity for bidirectional regulation. Memory consolidation theory suggests that memory is maintained at a stable strength after formation. The authors show that memory is dynamically maintained at an intermediate level allowing a bidirectional regulation which is mediated by a balance between activated Rac1 and expressed α2-chimaerin. Guided by such widely accepted theory, the study of memory maintenance is understandably devoted to the identification of learning-stimulated self-sustainable protein synthesis regulatory mechanisms5. Indeed, a number of candidate mechanisms for such a simple maintenance have been reported, including the autophosphorylation of calcium/calmodulin-dependent protein-kinase type II (CAMKII)7, and the persistent protein synthesis regulated via self-perpetuating prion-like translational factors such as the cytoplasmic polyadenylation binding protein-3 (CPEB-3)8. It is a common experience, however, when recalling an episode that happened some time ago, the memory is vivid at one time but vague at others. Such flexible recalling experiences seem to be counterintuitive to memory consolidation theory. In addition to common experiences, it is noteworthy that recent publications have reported a number of intriguing observations that appear inconsistent with the notion of stabilized memory. First, synaptic connections involved in maintaining LTM are highly dynamic5. Second, overexpression of the self-autonomous protein-kinase-M-zeta (PKMζ)9 was reported to enhance memory long after memory formation, but mechanisms underlying such enhancement remain unexplained10.Memory consolidation theory suggests that a newly acquired labile memory is gradually consolidated or stabilized through gene-regulation-based persistent modifications in synaptic structures within neural circuits and the resulting consolidated long-term memory (LTM) is maintained at a stable strength11. The recently proposed concept of active forgetting describes an intrinsic mechanism, i.e., learning or training evokes active forgetting to accelerate the decay of a formed memory. Multiple mechanisms are attributed to forgetting, including through activation of Rac1 or Cdc42, as well as through neurogenesis11. Recent progress in study of forgetting reveals that suppression of forgetting, inhibition of Rac1-dependent forgetting in particular, is capable of prolonging an hour-long labile memory to days14 in a range of tasks from invertebrates to vertebrates16. Moreover, a study reports that LTM decayed away during infancy can be recovered in adulthood17, suggesting that neurogenesis-based forgetting does not lead to memory erasure. These observations got us interested in investigating whether forgetting mechanisms, Rac1-dependent forgetting in particular, play any roles in memory maintenance. One possible candidate molecule to inhibit Rac1 activity comes from recent studies in mice during developmental stages21. It reveals that the negative regulator of Rac1 activity, α2-chimaerin, acts as a brake through its GTPase-activating function to constrain Rac1 activity in regulating hippocampal dendrite and spine morphogenesis22 for establishment of normal cognitive ability23. However, the roles of interaction between Rac1 activity and α2-chimaerin in memory maintenance in adulthood remain to be unraveled.Forgetting, the flip side of memory consolidation, is also an important feature for different types of memoriesThe present study provides a novel mechanism for dynamic memory maintenance that affords an intuitive explanation as to why LTM is still capable of being enhanced or faded long after completing formation. By integrating genetic, pharmacological, and optogenetic manipulations during behavioral paradigms, we demonstrate that the interplay between learning-evoked Rac1-dependent forgetting and learning-induced expression of a memory molecule α2-chimaerin enables consolidated contextual fear memory and is maintained at an intermediate level so that memory could be enhanced through expression of more α2-chimaerin while attenuated through elevated Rac1 activity. Moreover, forgetting induced by enhanced Rac1 activity is reversible through suppression of Rac1 activity. Thus, our results reveal that consolidated memory is maintained through the learning-evoked Rac1-dependent reversible forgetting mechanism balanced by learning-evoked expression of a memory maintenance molecule, α2-chimaerin.14, we were interested in studying whether Rac1-dependent forgetting mechanisms play roles in LTM maintenance. In an effort to investigate this issue, we assayed Rac1 activity in hippocampal extracts of mice trained in the contextual fear conditioning (CFC) during the maintenance period of memory. Wild-type (WT) mice were subjected to single- or three-trial CFC and were tested for memory retention at different time points   Fig. . To avoiays Fig.  and thenays Fig. . Consideays Fig. . Consistays Fig. . In addiays Fig. . Howeverays Fig. .Fig. 1In15. Mice were bilaterally injected with adeno-associated viruses (AAVs), carrying mutant transgenes that encoded either dominant-negative Rac1 (Rac1-DN) to inhibit endogenous Rac1 activity or constitutively active Rac1 (Rac1-CA) to increase Rac1 activity, targeted to hippocampal excitatory neurons15  and AAV9-tetracycline response element (TRE)-Tandem dimer Tomato (tdTomato) to the CA1 region of WT mice  was assessed by the percentage of Rac1-GTP+/c-fos+ colabeled cells among c-fos+ cells (engram cells). We found that 79% of c-fos+ cells were labeled with activated Rac1 positive (Rac1-GTP+) at 24 h following training  in CA1 engram cells and the control (tdTomato) mice were injected with AAV9-c-fos:tTA and AAV9-TRE-tdTomato  mice were injected bilaterally into the CA1 with a virus cocktail of AAVato Fig. , top. Inato Fig. , bottom.ato Fig.  at day 4ato Fig. . Next, wThe findings from the above two experiments led us to make a third prediction that the naturally faded memory for several days . Additional injections of ANI further supported the finding Fig. . This ex23. For knockdown, we generated AAVs that carry a short hairpin RNA (shRNA) sequence22 that targets α2-chimaerin (AAV-α2 shRNA), fused to tdTomato, under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter  induced via a single tetanus (weak stimulation) at the Schaffer collaterals of the hippocampal CA1 region. Knockdown of α2-chimaerin in slices expressing AAV-α2 shRNA led to a reduction of potentiation and accelerated LTP decay Fig. . In cont 14 Fig. , because 14 Fig.  while ac 14 Fig. .Fig. 6α215. Thus, interplay between activated Rac1 and expressed α2-chimaerin defines the intermediate level of memory during maintenance period and allows dynamic memory maintenance  were purchased from the Vital River Laboratory  and were housed in groups under standard conditions according to the Tsinghua University animal facility. Mice were maintained on a 12 h light/dark cycle and were tested during the light phase of the cycle. All animal work complied with ethical regulations for animal testing and research, and was done in accordance with Institutional Animal Care and Use Committee (IACUC) approval by Tsinghua University and followed by all Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.All plasmids were constructed by standard molecular biology procedures and subsequently verified by double-strand DNA sequencing. The α1-chimaerin and α2-chimaerin were synthesized by the GENEWIZ company (sequence are showed below). The pAAV-CaMKIIα-EGFP was a gift from Bryan Roth (Addgene plasmid #50469). The α1-chimaerin and α2-chimaerin were sub-cloned into AAV backbone pAAV-CaMKIIα-EGFP using the BsrGI/EcoRI restriction enzymes.α1-chimaerin (Sequence ID: NM_001206602.1)5′-ATGCCATCCAAAGAGTCTTGGTCAGGGAGGAAAACTAATAGGGCTGCAGTTCACAAATCAAAACAAGAGGGCCGTCAGCAAGATTTATTGATAGCAGCCTTGGGAATGAAACTGGGTTCTCCAAAGTCGTCTGTGACAATCTGGCAACCTCTGAAACTCTTTGCTTATTCGCAGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3′α2-chimaerin (Sequence ID: NM_001822.5)5′-ATGGCCCTGACCCTGTTTGATACAGATGAATATAGACCTCCTGTTTGGAAATCTTACTTATATCAGCTACAACAGGAAGCCCCTCATCCTCGAAGAATTACCTGTACTTGCGAGGTGGAAAACAGACCAAAGTATTATGGAAGAGAGTTTCATGGCATGATCTCCAGAGAAGCAGCCGACCAGCTCTTGATTGTGGCTGAGGGGAGCTACCTCATCCGGGAGAGCCAGCGGCAGCCAGGGACCTACACTTTGGCTTTAAGATTTGGAAGTCAAACCAGAAACTTCAGGCTCTACTACGATGGCAAGCACTTTGTTGGGGAGAAACGCTTTGAGTCCATCCACGATCTGGTGACTGATGGCTTGATTACTCTCTATATTGAAACCAAGGCAGCAGAATACATTGCCAAGATGACGATAAACCCAATTTATGAGCACGTAGGATACACAACCTTAAACAGAGAGCCAGCATACAAAAAACATATGCCAGTCCTGAAAGAGACACATGATGAGAGAGATTCTACAGGCCAGGATGGGGTGTCAGAGAAAAGGTTGACATCACTTGTTAGAAGAGCAACTCTGAAAGAAAACGAGCAAATTCCAAAATATGAAAAGATTCACAATTTCAAGGTGCATACATTCAGAGGGCCACACTGGTGTGAATACTGTGCCAACTTTATGTGGGGTCTCATTGCTCAGGGAGTGAAATGTGCAGATTGTGGTTTGAATGTTCATAAGCAGTGTTCCAAGATGGTCCCAAATGACTGTAAGCCAGACTTGAAGCATGTCAAAAAGGTCTACAGCTGTGACCTTACGACGCTCGTGAAAGCACATACCACTAAGCGGCCAATGGTGGTAGACATGTGCATCAGGGAGATTGAGTCTAGAGGTCTTAATTCTGAAGGACTATACCGAGTATCAGGATTTAGTGACCTAATTGAAGATGTCAAGATGGCTTTCGACAGAGATGGTGAGAAGGCAGATATTTCTGTGAACATGTATGAAGATATCAACATTATCACTGGTGCACTTAAACTGTACTTCAGGGATTTGCCAATTCCACTCATTACATATGATGCCTACCCTAAGTTTATAGAATCTGCCAAAATTATGGATCCGGATGAGCAATTGGAAACCCTTCATGAAGCACTGAAACTACTGCCACCTGCTCACTGCGAAACCCTCCGGTACCTCATGGCACATCTAAAGAGAGTGACCCTCCACGAAAAGGAGAATCTTATGAATGCAGAGAACCTTGGAATCGTCTTTGGACCCACCCTTATGAGATCTCCAGAACTAGACGCCATGGCTGCATTGAATGATATACGGTATCAGAGACTGGTGGTGGAGCTGCTTATCAAAAACGAAGACATTTTATTTTAA-3′α1-chimaerin was 5′-GCTT TCAGCAATGTGTCAT-3′39. The α2-chimaerin shRNA sequence binding to the mouse α2-chimaerin was 5′-GCACATGGCAGTCCTGAAA-3′, whereas the negative control scrambled shRNA (ctrl shRNA) was 5′-GTTCATATGTTCACCTATT-3′22. The U6 promoter sequence was 5′- GATCCGACGCCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGATACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTT-3′.The U6 promoter-driven α1-chimaerin shRNA (α1 shRNA), α2-chimaerin shRNA (α2 shRNA), or negative control scrambled shRNA (ctrl shRNA) constructs were synthesized (GENWIZ company) and sub-cloned into AAV backbone pAVV-CAG-tdTomato (Addgene plasmid # 59462) using the EcoRI/HindIII and EcoRV/HindIII restriction enzymes, respectively. The α1-chimaerin shRNA sequence binding to the mouse 39. Keyhole limpet hemocyanin-coupled synthetic peptides for α1-chimaerin was MPSKESWSGRKANR and keyhole limpet hemocyanin-coupled synthetic peptides for α2-chimaerin was HDEKEATGQDGVSEKR. Anti-Rac1-GTP (active Rac1) monoclonal antibody was from the NewEast company (Cat. No. 26903). Anti-Rac1 antibody was from BD Transduction Laboratories (Cat. No. 610650). Anti-phospho-PAK1/2/3 antibody was from novusbio company (Cat. No. NB100-82131). Anti-Cleaved Caspase-3 antibody was from Cell Signaling (Cat. No. 9661). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Cat. No. #7072) and HRP-conjugated goat anti-rabbit IgG (Cat. No. #7071) were from Cell Signaling Technology. Alexa Fluro® 488 affinipure donkey anti-mouse IgM was from the Jackson ImmunoResearch Laboratories company (Cat. No. #711-545-140). Actin antibody was from the Merck Millipore company (Cat. No. #MAB1501).The following antibodies were used: primary antibodies for α1-chimaerin and α2-chimaerin were raised in rabbits immunized with keyhole limpet hemocyanin-coupled synthetic peptides as previously reported15. ANI  was dissolved in 0.9% saline and the pH was adjusted with 1 N HCl to 7.0–7.4. Mice were subcutaneously injected with 150 mg per kg of ANI or an equivalent volume of saline immediately after training. This amount of ANI was shown to effectively inhibit cerebral protein synthesis in mice (~96%)40.Ehop016  was dissolved in a solution containing 1% dimethylsulfoxide/30% PEG/1% Tween-80. Mice were intraperitoneally injected with 20 mg per kg of Ehop016 solution or an equivalent volume of saline. At the dose used, Ehop016 has high efficiency to block Rac1 activity9-c-fos:tTA (300 nl of AAV9-c-fos:tTA) and 300 nl of the AAV9-TRE-tdTomato into the CA1 . For the activation of Rac1 activity in CA1 engram cells, a virus cocktail AAV9-c-fos:tTA (300 nl of AAV9-c-fos:tTA and 300 nl of AAV9-TRE:photoactivatable Rac1 (PaRac1)-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) were injected into the mice CA1 . Following the injections, the needle stayed for 10 min before slowly withdrawn and the wound sutured. After surgery, animals were allowed to recover for two weeks prior to the performance of all subsequent experiments.Mice were anesthetized with 0.2% sodium pentobarbital (5 ml/kg) and fixed to a stereotaxic frame. Their body temperature was kept at 36 °C by a heating pad and their skull was exposed. Furthermore, holes in the skull were drilled by a micromotor drill. The injection of the virus was performed using a 10 ml nanofil syringe under controlled by the UMP3 and Micro4 system (WPI), with a speed of 50 nl/min. The dorsal hippocampus injections were bilaterally targeted to −2.0 mm AP, ±1.5 mm ML, and −1.5 mm DV; the DG injections were bilaterally targeted to -2.0 mm AP, ±1.5 mm ML, and −2.0 mm DV. The viral volumes of the AAV-Rac1-CA, AAV-Rac1-DN, and AAV-control were 400 nl for DG and 600 nl for CA1. Mice were bilaterally injected with 1 µl of either AAV-α1-chimaerin, AAV-α2-chimaerin, AAV-control, AAV-α1-chimaerin shRNA, AAV-α2-chimaerin shRNA, or AAV-control shRNA. For labeling of the CA1 engram cells, a virus cocktail AAV9-c-fos:tTA) and 500 nl of the AAV9-TRE:PaRac1-tdTomato41 or the AAV9-c-fos-EGFP (as the control group) into the CA1. Furthermore, they were bilaterally implanted with optical fibers  into the CA1 . Mice were allowed to recover for two weeks under the ON-Dox condition prior to the start of the behavioral experiments. The cohorts of mice used for the optogenetic engram stimulation, mice were fed regular food, without Dox, for 24–30 h until contextual fear-conditioning training. After CFC, mice were returned to their home cage under the ON-Dox condition. Following behavioral testing, brain sections were examined to confirm efficient virus-mediated labeling of the target areas.Mice were bilaterally injected with a virus cocktail (500 nl of AAV2) was measured at the tip of the fiber. A blue light from a laser was ON for 30 s to stimulate the photoactivatable Rac1 for five trials, with trial intervals of 120 s31. For all behavioral procedures, the freezing behavior was measured in light stimulated mice at 1.5 h after stimulation. Successively, animals were put back to their home cage.Mice were intra-CA1 implanted with optic fiber after AAV injection. The optic fibers of mice were connected to a 473 nm blue laser diode via a FC/PC adaptor. Light intensity  had a stainless-steel rod floor which was connected to a shock generator in a sound-attenuating box. In single-trial CFC sessions, mice were individually placed in the conditioning and freely explored the area for 3 min. Then, mice were exposed to one footshock  and were returned to their home cage 30 s after. In the three-trial CFC task, the footshock  was delivered at 180, 240, and 300 s. Mice remained in the conditioning chamber for a total of 330 s. In only context or only shock training, mice were exposed in a context for 3 min without shock or one footshock  in the absence of context. After that, mice were placed back in their home cage. During testing, mice were placed back in the conditioning chamber for 4 min at different times . For all procedures, the animal freezing behaviors were monitored using a manufacturer’s software.The single- and three-trial CFC procedures were based on those of Denny et al.Subject mice were individually placed in the three-chamber apparatus for 10 min to habituate to the environment (day 1). On day 2, a 6-week-old male C57BL/6J mouse was placed in the left grid enclosure for familiarization. Successively, the test mouse was located in the center compartment of the three-chamber apparatus for 10-min social training. During experimental testing (1 h and day 3), a novel 6-week-old male C57BL/6J mouse was placed in the right grid enclosure and the familiar mouse in the left grid enclosure. And then, test mouse was placed into the social chamber and was allowed to explore for 10 min. The amount of time for the test mouse spent in close interaction was recorded using the Any maze system. The discrimination index (DI) was calculated using the following formula: (time exploring the novel mouse − time exploring the familiar mouse)/(time exploring the novel mouse + time exploring the familiar mouse) * 100.15. For the testing phase, one of the objects was exchanged with a new one, and the time spent exploring the two objects was separately recorded using ANY-MAZE software. The DI was calculated using the following formula: (time exploring the novel object − time exploring the familiar object)/(time exploring the novel object + time exploring the familiar object) × 100. For the memory decay curve, time intervals between sampling and testing were 1 and 24 h.For the habituation phase, individual adult male mice were placed in a chamber (50 cm × 50 cm × 40 cm) and allowed to freely explore the context for 10 min while being recorded by an overhead camera. This phase was also conducted as the open field test to assess the animal’s basal locomotor activity indicated by the average speed and anxiety levels indicated by the percentage of time spent in the middle arena (30 cm × 30 cm). For the sampling phase, the animal was placed in the same chamber containing two different objects for 5 min and allowed to explore the object. To saturate the odor left by the previous mice and to make the mice more relaxed, we placed standard animal beddings in the chamberIsolated hippocampi were homogenized in cell lysis buffer  with protease inhibitors. The protein concentration was determined using the BCA protein assay kit . For the detection of the levels of α1-chimaerin, α2-chimaerin, total Rac1 and actin, 20 μg of homogenates was separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . Membranes were blocked in milk solution (5% milk in TBS and 0.1% Tween 20) for one hour at room temperature. Subsequently, membranes were individually incubated with primary antibodies against α1-chimaerin , α2-chimaerin , total Rac1 , and actin  overnight at 4 °C. All the HRP-conjugated secondary antibodies were used at 1:2000 dilutions for membranes incubation at room temperature for one hour. For the relative level of the Rac-GTP assay, a GST-tagged PAK-PBD beads  were incubated with the lysate mix (300 μg) for overnight at 4 °C. The beads were washed three times with lysis buffer at room temperature. Then, bound proteins were eluted with loading buffer  and analyzed by western blotting. Image quantification analysis in bands of the western blots was calculated by the ImageJ software .Mice were anesthetized with 0.2% sodium pentobarbital (5 ml/kg) and perfused intracardially with 30 ml of 4% paraformaldehyde. The brains were fixed in 4% paraformaldehyde overnight at 4 °C and serial coronal sections (30 μm) were then taken throughout the hippocampus using a vibratome. The floating sections were collected and rinsed in a 0.1 M phosphate-buffered saline (PBS) (pH 7.4) solution with 0.1% Triton X-100 and incubated with blocking solution (10% donkey serum and 0.1% Triton X-100) for 2 h at room temperature. The slices were then incubated with the following primary antibodies at 4 °C for overnight: anti-active Rac1 , anti-Cleaved Caspase-3 , and anti-phospho-PAK1/2/3 . Following three times rinsed (30 min each) in the PBS solution, slices were incubated with the secondary antibodies conjugated with dyes. Then sections were washed three times with PBS solution and mounted on slides with antifade mounting medium . The images of the immunohistochemistry were captured on a Zeiss LSM 710.Cell counting of c-Fos-positive cells and Rac1-active cells was performed in the CA1 by utilizing imaging analysis function of Zeiss software (Zen blue 2.3). The quantification of the number of c-Fos-positive cells was performed by thresholding c-Fos immunoreactivity above background levels automatically. The quantification of the number of Rac1-GTP cells was performed by setting the threshold manually from 1000 to 4096. The quantification of the number of phospho-PAK cells was performed by setting the threshold manually from 692 to 4096. The quantification of the number of cells with 4′,6-diamidino-2-phenylindole was performed by setting the threshold manually from 616 to 4096.2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose) for at least 1 h. Following the recovering at room temperature, the slices were transferred to the field excitatory post-synaptic potentials (EPSPs) recording chamber  with ACSF (flow rate 1.5 ml/min) for at least 20 min prior to the recording. Filed EPSPs were evoked from CA3-CA1 synapses through the stimulation of the Schaffer collateral axons and recorded in CA1 extracellular fEPSPs. The stimulation intensity was adjusted to elicit a 30% maximal response. The LTP protocol for the 100 Hz consisted in 10 × 100 Hz bursts (four pulses per burst) with a 200 ms interval between bursts. Data acquisition and analysis were performed using the multielectrode MED64 hardware and software packages (Panasonic).Transverse hippocampal slices were prepared from 4-month-old mice. Animals were killed by decapitation in accordance to the institutional regulations. Hippocampi were sliced (300 μm) by a VF-300 microtome (Precisionary Instruments) and kept in an interface chamber with oxygenated artificial cerebrospinal fluid  or two-way ANOVA where appropriate. The data are shown as the mean ± SEM and NS indicates non-significance (p > 0.05). The significant levels were set to P = 0.05. Significant for comparison: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The specific statistical tests used and the details of p-values for each experiment can be found in Supplementary Table Statistical analyses were performed in GraphPad Prism. All data were analyzed with an unpaired Further information on research design is available in the Supplementary InformationTransparent Peer Review FileReporting Summary


Correction to: Nature Communications 10.1038/s41467-018-04901-6; published online 27 June 2018In the original version of the Supplementary Information file associated with this Article, the sequence ‘1x MS2 scRNA.b2’ was incorrectly given as ‘GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCGCACATGAGGATCACCCATGTGCTTTTTT’ and should have read ‘GAAGATCCGGCCTGCAGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACATGAGGATCACCCATGTGCTTTTTTT’. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.


Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics. Protein nanopores are emerging as sensors for a variety of biomolecules. Here the authors develop a nanopore based on the bacterial toxin ClyA, in conjunction with binding proteins for glucose and asparagine, to detect these biomolecules simultaneously from a variety of unprocessed, diluted body fluids. The ability to monitor the health of individuals that exchange information through the Internet without requiring human intervention would undoubtedly bring about a revolution in personalised healthcare. Over the past decades, the miniaturisation of electronic devices combined with recent advances in the wireless transmission of data and microfluidics have opened up new possibilities for making such devices. However, one key area that is preventing their breakthrough is the production of sensing elements for biologically relevant molecules, which can be easily interfaced with silicon-based electronics. Such elements have long lagged behind the electronic industry in terms of miniaturisation and cost reduction.1. However, such oxidase enzymes are not ubiquitous, and today’s commercially available home diagnostic and implantable devices are entirely limited to one well-known example: the glucose sensor. However, despite the sensor has seen many improvements since it was first introduced in the early 1960s2, it still suffers from limitations including enzyme instability, complicated surface immobilisation procedures, baseline shifts, and does not tolerate well changes in micro-environmental factors such as changes in pH and ionic strength5.At the moment, biosensors that can be integrated in electronic devices rely mostly on the electric signal that is generated by the oxidation of a target analyte catalysed by an immobilised enzyme6. Nanopores can also detect polypeptide8 biomarkers. However, their use in diagnostic devices is complicated by the low, typically picomolar, concentration of such biomarkers in biological fluids. By contrast, metabolites are highly abundant in bodily fluids, and their variation in concentration is linked to many diseases9. Nanopores equipped with cyclodextrins10, cucurbiturils11, or cyclic peptides12 may recognise metabolites. However, the detection and quantification of such analytes directly from a biological sample cannot be easily implemented because the recognition elements lack selectivity.Nanopores, which are nanometre-sized water conduits spanning biological or artificial membranes, are emerging as a new class of biosensors. When a potential is applied, the flux of ions across an individual nanopore generates an electrical signal that allows direct interfacing with electronic devices. Most notably, nanopores can be integrated into electronic devices and are now used for low-cost, high-speed, and portable sequencing of DNA16. Here, using proteins from a protein family that in cells recognise an enormous variety of molecules, we show that ClyA nanopores can report the concentration of glucose and asparagine directly from samples of blood, sweat, and other bodily fluids. Conveniently, no sample preparation is required and the concentration of the metabolite can be monitored continuously.Recently, we have shown that the biological nanopore cytolysin A (ClyA) can be used to monitor the function of proteins when they are lodged inside the nanopore Fig. 13–16. He17. GBP belongs to the class of substrate-binding proteins which, with minor structural differences, recognise hundreds of different metabolites in cells18. The structure of the GBP has been solved in the ligand-free state (open or apo form) configuration and in the closed, ligand-bound, configuration. Interestingly, NMR studies revealed that in solution, apo-GBP adopts both configurations even in the absence of glucose19. In our experimental setup, GBP (44 nM) was added to the cis side of a ClyA-AS20 nanopore . Two current levels 19.In this work, we used a glucose-binding protein (GBP) that binds glucose with high affinity and specificitytrans side of the nanopore increased the number of events corresponding to the closed configuration  and opening  rate constants could be determined from the inverse of L1 and L2 dwell times 21. The dependence of the fractional time of GBP in the closed configuration with the glucose concentration fitted well to a Hill function with the Hill coefficient set to one  corresponded well to the value measured in bulk (2–5 × 10−7 M)23. A GBP variant (GBP-A213R) with reduced affinity for glucose25 showed only one single level in the absence of glucose . Extrapolation from the calibration curve , sweat (2 µL), urine (200 nL), and saliva (8 µL) were added to the rve Fig.  allowed 0 nL, swe26, while its abnormal concentration in blood has been associated to Parkinson’s disease27. Under −90 mV, the blocked pore level of apo-SBD1 contained one main current level corresponding to the open protein configuration . As observed before for GBP, the binding constants measured by the nanopore were similar to the ones measured in bulk28. The percentage of bound fraction was used to extrapolate the concentration of asparagine in the trans solution  was markedly different than the open protein configuration of GBP (Ires% = 69.1 ± 0.2%). Thus, when GBP (44 nM) and SBD1 (120 nM) were simultaneously present in the cis solution, blockades corresponding to each protein adaptor could be readily distinguished , enzymes from Thermo Fisher Scientific, and lipids from Avanti Polar Lipids.Escherichia coli at the beginning of the gene . To maintain the open-reading frame, two extra bases were inserted after the NcoI site, resulting in an additional alanine residue after the starting methionine. For purification, a His6-tag was attached at the C-terminus of GBP, via a flexible glycine–serine–serine linker and the open-reading frame was terminated by two consecutive stop codons, followed by a Hind III restriction site (3′-end). The attachment of the His6-tag was carried out in two consecutive PCR reactions. During the first PCR reaction, the GBP gene was amplified directly from a single BL21 (DE3) E. coli (Lucigen) colony using Phire Hot Start II DNA polymerase, 0.4 µM dNTPs, 6 µM of MS-Glu_F (TATATATCCATGGCTaataagaaggtgataaccctgtctgctgtg), and MS-Glu_r1 (gatggtgatgGCTGCTGCCtttcttgctgaattcagccaggttgtctttatctacgcc) primers in a 50 µL reaction volume. The PCR reaction cycling protocol was as follows: pre-incubation step at 98 °C for 30 s and then 30 cycles of denaturation at 98 °C for 5 s and extension at 72 °C for 1 min. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen) and served as a template for the second PCR reaction, which used ~100 ng of the purified PCR product amplified by Phire Hot Start II DNA polymerase using 0.4 µM dNTPs, 6 µM of MS-Glu_F, and MS-Glu_r2 (atatatataagctttcattaatggtGATGGTGATGGCTGCTGCCTTTCTTGCTGAATTC) primers in 300 µL volume. The cycling protocol was the same as in the previous step. The resulting PCR product encoding for the His6-tagged GBP gene was purified with QIAquick PCR Purification Kit (Qiagen) and digested with NcoI and HindIII . The gel purified insert  was cloned under control of the T7 promoter into the pT7-SC1 expression plasmid29 using sticky-end ligation  via NcoI (5′) and HindIII (3′) sites. Ligation mixture of 0.6 µL was transformed into 50 µL of E. cloni® 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37 °C on ampicillin-containing (100 µg/mL) LB agar plates. The identity of the clones was confirmed by sequencing.To allow cloning, a NcoI site (CCATGG) was introduced into the sequence of the wild-type GBP from MANKKVITLSAVMASMLFGAAAHAADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKKGSSHHHHHCCATGGCTAATAAGAAGGTGATAACCCTGTCTGCTGTGATGGCCAGCATGTTATTCGGTGCCGCTGCACACGCTGCTGATACTCGCATTGGTGTAACAATCTATAAGTACGACGATAACTTTATGTCTGTAGTGCGCAAGGCTATTGAGCAAGATGCGAAAGCCGCGCCAGATGTTCAGCTGCTGATGAATGATTCTCAGAATGACCAGTCCAAGCAGAACGATCAGATCGACGTATTGCTGGCGAAAGGGGTGAAGGCACTGGCAATCAACCTGGTTGACCCGGCAGCTGCGGGTACGGTGATTGAGAAAGCGCGTGGGCAAAACGTGCCGGTGGTTTTCTTCAACAAAGAACCGTCTCGTAAGGCGCTGGATAGCTACGACAAAGCCTACTACGTTGGCACTGACTCCAAAGAGTCCGGCATTATTCAAGGCGATTTGATTGCTAAACACTGGGCGGCGAATCAGGGTTGGGATCTGAACAAAGACGGTCAGATTCAGTTCGTACTGCTGAAAGGTGAACCGGGCCATCCGGATGCAGAAGCACGTACCACTTACGTGATTAAAGAATTGAACGATAAAGGCATCAAAACTGAACAGTTACAGTTAGATACCGCAATGTGGGACACCGCTCAGGCGAAAGATAAGATGGACGCCTGGCTGTCTGGCCCGAACGCCAACAAAATCGAAGTGGTTATCGCCAACAACGATGCGATGGCAATGGGCGCGGTTGAAGCGCTGAAAGCACACAACAAGTCCAGCATTCCGGTGTTTGGCGTCGATGCGCTGCCAGAAGCGCTGGCGCTGGTGAAATCCGGTGCACTGGCGGGCACCGTACTGAACGATGCTAACAACCAGGCGAAAGCGACCTTTGATCTGGCGAAAAACCTGGCCGATGGTAAAGGTGCGGCTGATGGCACCAACTGGAAAATCGACAACAAAGTGGTCCGCGTACCTTATGTTGGCGTAGATAAAGACAACCTGGCTGAATTCAGCAAGAAAGGCAGCAGCCATCACCATCACCATTAATGAAAGCTT30 on a circular plasmid template DNA (pT7-SC1 containing GBP gene) with Phire® Hot Start Polymerase (Finnzymes) by using two complementary primers. Six µM forward (GGTTATCGCCAACAACGATAGGATGGCA) and 6 µM reverse primer (GCGCCCATTGCCATCCTATCGTTGTT) containing a codon encoding the mutation were used in 50 µL final volume to amplify the gene . The PCR product was incubated with DpnI (1 FDU) for 1 h at 37 °C to digest the plasmid template. The remaining DpnI enzyme was deactivated at 55 °C. The mixture was then incorporated into E. cloni® 10G cells (Lucigen) by electroporation. Transformants containing the plasmid were grown overnight at 37 °C on LB agar plates supplemented with 100 μg/mL ampicillin and 1% glucose. Single colonies were picked and inoculated into 10 mL LB medium supplemented with 100 mg/L of ampicillin for plasmid DNA preparation. The presence of the mutation was confirmed by sequencing of the plasmid.The GBP-A213R was constructed using the QuickChange protocol for site-directed mutagenesisE. coli strain MC1061 and purified as described previously.28 The cell lysate was mixed with 50 mM potassium phosphate, pH 8.0, 200 mM KCl, 20% glycerol (buffer A) supplied with 20 mM imidazole and incubated with Ni2+-sepharose  resin for 1 h at 4 °C. Next, the resin was washed with 20 column volumes of buffer A supplied with 50 mM imidazole, followed by protein elution with 500 mM imidazole in buffer A. To prevent protein aggregation, 5 mM EDTA was added immediately after elution. Afterwards the histidine-tag was cleaved off by adding 2.5% w/w His-TEV (S219V)31 and dialyzing overnight against 50 mM Tris-Cl, 0.5 mM DTT, 0.5 mM EDTA, pH 8.0 to remove imidazole. The histidine-tagged tobacco etch virus (TEV) protease and residual uncut protein were removed by loading it on a small 0.5 mL Ni2+-sepharose column collecting the flow-through. Next, proteins were further purified with size-exclusion chromatography on a Superdex-200 column  equilibrated with 50 mM Tris-HCl, 200 mM NaCl, pH 7.5. Peak fractions were collected in aliquots and were stored at −80 °C after flash-freezing in liquid nitrogen.Substrate-binding domains carrying a histidine-tag were expressed in E. cloni® EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the ClyA-AS gene. ClyA-AS contains eight mutations relative to the S. Typhi ClyA-WT: C87A, L99Q, E103G, F166Y, I203V, C285S, K294R, and H307Y. Transformants were selected after overnight growth at 37 °C on LB agar plates supplemented with 100 mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400 mL 2xYT medium containing 100 mg/L of ampicillin. The cells were grown at 37 °C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of ClyA-AS was induced by addition of 0.5 mM IPTG. The cell cultures were further grown overnight at 25 °C. The next day, the bacteria were harvested by centrifugation at 6000×g at 4 °C for 25 min and the pellets were stored at −80 °C. The pellets containing monomeric ClyA-AS were thawed and solubilized with 20 mL lysis buffer  and incubated for 20 min at 37 °C. The bacteria were then lysed by sonication (Branson). The lysate was subsequently centrifuged at 6000×g at 4 °C for 20 min and the cellular debris discarded. The supernatant was mixed with 150 μL of Ni-NTA resin (Qiagen) in wash buffer . After 1 h, the resin was loaded into a column  and washed with ~5 mL of the wash buffer. The protein was eluted with ~100 μL elution buffer . Type I ClyA-AS oligomers were obtained by incubation of ClyA-AS monomers with 0.2% β-dodecylmaltoside (DDM) for 30 min at 37 °C. The oligomers were separated from monomers by blue native polyacrylamide gel electrophoresis  using 4–20% polyacrylamide gels20. The band corresponding to type I ClyA-AS were cut out from the gel and was extracted in 150 mM NaCl, 15 mM Tris-HCl, pH 7.5 supplemented with 0.2% DDM and 10 mM EDTA.E. cloni® EXPRESS BL21 (DE3) cells were transformed with the pT7-SC1 plasmid containing the His6-tagged GBP gene. Transformants were selected after overnight growth at 37 °C on LB agar plates supplemented with 100 mg/L ampicillin and 1% glucose. The resulting colonies were inoculated into 400 mL 2xYT medium containing 100 mg/L of ampicillin. The cells were grown at 37 °C (200 r.p.m. shaking) until an OD600 of ~0.8 was reached. The expression of GBP was induced by addition of 0.5 mM IPTG. The cell cultures were further grown overnight at 25 °C. The next day, the bacteria were harvested by centrifugation at 6000×g at 4 °C for 25 min. The protein was purified directly afterwards by osmotic shock. The pellets were thoroughly dissolved in 20% ice-cold sucrose solution and incubated, while shaking at 4 °C for 10 min. The cell suspension was centrifuged at 6000×g at 4 °C for 10 min and cells collected. The cells were re-suspended in 4× volume of ice-cold MilliQ water supplemented with 5 mM MgCl2 and incubated, while shaking, on ice for 20 min. The cells collected again by centrifugation at 6000×g at 4 °C for 20 min. The supernatant was mixed with 150 μL of Ni-NTA resin (Qiagen) in wash buffer . After 1 h, the resin was loaded into a column  and washed with ~5 mL of the wash buffer. The protein was eluted with ~500 μL elution buffer . The protein (500 μL) was then dialysed exhaustively overnight against dialysis buffer , whereby the dialysis buffer was replaced for fresh buffer at least three times. Protein concentration was measured by using the Nanodrop 2000C at 280 nm (Thermo Fisher Scientific). Protein was stored with 10% glycerol in −80 °C after flash-freezing until further use.sn-glycero-3-phosphocholine (DPhPC) in pentane (10 mg/mL) was then added to the buffer  present in both compartments. After evaporation of the pentane, a lipid monolayer at the air–water interface can self-assemble spontaneously by lowering and raising the buffer across the aperture. 0.01–0.1 ng of purified ClyA-AS were added to the grounded cis compartment. The reconstitution of single nanopores was monitored electrically by applying a −35 mV bias. ClyA-AS nanopores display a higher open pore current at positive than at negative applied potentials, which provides a useful tool to determine the orientation of the pore. After insertion of a single pore, GBP or SBD1 were added to the cis compartment. This was followed by addition of a glucose or asparagine sample or bodily fluid sample in the trans compartment.The setup consisted of two chambers separated by a 25 μm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an aperture of approximately 100 μm in diameter. The aperture was pre-treated with a solution of hexadecane [10% (v/v) in pentane] to leave a hydrophobic coating to support bilayer formation. 1,2-diphytanoyl-All traces were routinely recorded at −90 mV or −60 mV applied transmembrane potential by using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitised with a DigiData 1440 A/D converter (Axon Instruments). The data was sampled by applying a 2 kHz low-pass Bessel filter and at a frequency of 10 kHz. Obtained traces were filtered digitally with a Gaussian low-pass filter with a 100 Hz cutoff. Data were recorded by using the Clampex 10.4 software (Molecular Devices) and the subsequent analysis was carried out with the Clampfit software (Molecular Devices).τon) and ligand-independent opening (τoff) of GBP were obtained from single exponential fits to histograms of the inter-event times and dwell times, respectively. The mean lifetime of the open state (τon) was converted to the on-rate constants by using kon = 1/τon[glucose], where [glucose] is the concentration of glucose added to the trans side. The τoff was converted to the off-rate by using koff = 1/τoff. The equilibrium binding constant was calculated by plotting the fraction of closed configuration against the concentration of substrate and the data fitted to a Hill function . Events shorter than 2 ms were ignored. The mean dwell times for the ligand-induced closing  were used to determine the glucose in the bodily fluids. The solution of the glucose assay kit contains a hexokinase enzyme that uses adenosine triphosphate (ATP) as a co-factor to phosphorylate glucose. The glucose-6-phosphate (G6P) is then oxidised to 6-phospho-gluconate in the presence of oxidised nicotinamide adenine dinucleotide (NAD) in a reaction catalysed by glucose-6-phosphate dehydrogenase (G6PD). The generation of NADH was spectrophotometrically observed by measuring absorbance at 340 nm. Under these conditions, the final concentration of produced NADH corresponds to the glucose concentration in the sample. In order to perform the experiment, a dilution of bodily fluid was added to the reaction mixture. The reaction was incubated for 15 min at room temperature and then the absorbance was measured at 340 nm (ε = 6220 M−1 cm−1). For each sample, three different dilutions were measured. The commercial glucose metre Accu-Chek® uses a mutant variant of quinoprotein glucose dehydrogenase and has a measurement range between 0.6 and 33.3 mmol/L32. The Accu-Chek® is considered to be a reliable device for monitoring glucose levels in blood from finger pricks33.A glucose assay kit (HK) from Sigma-Aldrich and a commercial glucose metre . The blood samples were collected shortly before the experiments to minimise ex vivo artefactual changes. Sweat, urine, and saliva samples were either freshly collected before, or stored at −20 °C, before the start of experiments. The solutions were kept on ice for the duration of an experiment. The blood samples were diluted 500-folds and the urine sample 10-folds with buffer  before adding 5 and 2 µL, respectively, to the kopening = 0.75 ± 0.12 s−1, 500 nM glucose), compared to the binding of asparagine to SBD1 , thus longer integration times are required for accurate reading. It follows that (GBP-A213R), which shows reduced affinity for glucose .All measurements were made by adding small volumes of biological samples to an electrophysiology chamber (500 µL). We did not observe a particular instability of the bilayer . After mixing, a concentrator  was used to evaporate all liquids by vacuum evaporation at 60 °C and 240×g, and the sample was reconstituted in an identical volume of water as the former fixed amount of supernatant. A final 5-min-long centrifugation at 20,000×g step was performed and the supernatant was stored at 4 °C before analysis.Proteins in 150 µL saliva and 60 µL serum samples were precipitated by incubation with 8% trichloroaceticacid (CASnr 76–03–9) for 5 min at 60 °C and 450 r.p.m. using a thermomixer  followed by cooling on ice for 30 min and centrifugation for 10 min at 20,000×HPLC quantification of asparagine was performed on an Agilent 1100 HPLC binary system  equipped with an 1100 Fluorescence detector (FLD) and a Gemini C18 column  while a 40 mM sodium phosphate buffer, pH 7.8 (solvent A) and a mixture of 45:45:10 acetonitrile/methanol/water (solvent B) were used. The fluorogenic labelling of amino acids was performed automatically by the auto sampler of the HPLC machine using a mixture of 10 mg o-phtalaldehyde  and 10 µL 3-mercaptapropionic acid  in water as reagent. The auto sampler mixed 0.5 µL sample with 1.3 µL 0.4 N borate buffer, pH 10.2 followed by 0.5 µL reagent and 7.6 µL water right before each injection. After the sample injection, amino acids were separated by applying solvent B at 1.5%/min increase for 25 min using a flow rate of 250 µL/min . Thereafter, the column was cleaned for 5 min at 66% solvent B followed by another 12 min at 100% solvent B. Then the column was equilibrated back to 5% solvent B. Fluorescence was measured at an excitation wavelength of 340 nm and an emission wavelength of 450 nm.Quantifications were performed using the method of standard addition at three different concentrations of analyte . The concentration of the analyte was obtained by measuring the peak areas, while each sample was analysed in triplicate. Citrulline was added as internal standard at 200 µM to correct for saliva and serum sample handling volume differences next to control run-to-run retention time shifts regarding all samples.Supplementary InformationPeer Review File