Engineering of specific bacteriophages for α-Synucleinopathies diagnosis

UMinho | 2022 Ad iana So ia Veloso Rod igues Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis Oc obe 2022 Ad iana So ia Veloso Rod igues Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis i Ad iana So ia Veloso Rod igues Enginee ing O Speci ic Bac e iophages Fo α- Synucleinopa hies Diagnosis Mas e Disse a ion Mas e ’s Deg ee in Biomedical Enginee ing Clinical Enginee ing B anch P ojec supe ised by Doc o I one Ma isa Pe ei a Ma ins Oc obe 2022 ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Es e é um abalho académico que pode se u ilizado po e cei os desde que espei adas as eg as e boas p á icas in e nacionalmen e acei es, no que conce ne aos di ei os de au o e di ei os conexos. Assim, o p esen e abalho pode se u ilizado nos e mos p e is os na licença abaixo indicada. Caso o u ilizado necessi e de pe missão pa a pode aze um uso do abalho em condições não p e is as no licenciamen o indicado, de e á con ac a o au o , a a és do Reposi ó iUM da Uni e sidade do Minho. Licença concedida aos u ilizado es des e abalho A ibuição-NãoCome cial-Compa ilhaIgual CC BY-NC-SA h ps://c ea i ecommons.o g/licenses/by-nc-sa/4.0/ iii AGRADECIMENTOS Dizem que odos nós somos um pedacinho de odas as pessoas que passam na nossa ida. De uma manei a ou de ou a, as pessoas deixam a sua pegada em nós e ans o mam-nos sem que às ezes nos ape cebamos disso. Sem dú ida que ao longo des a longa iagem passa am po mim pessoas que deixa am a sua ma ca, e o mínimo que lhes de o é um “ob igada”, po isso, as pala as que se seguem são dedicadas a elas. Emba quei nes e p oje o há um ano e com ela ap endi imenso… es e pa ág a o dedico-o à minha o ien ado a, I one Ma ins. Fo neces e-me odas as bases e supo e pa a le a es a missão a é ao im. Deixas e- me “c esce ” mos ando-me que e a az pa e e que o impo an e é en a e não desis i . Ob igada po udo… “You a e needed” o me. Dedico aqui ambém, um ag adecimen o ao A u Ribei o po odo o apoio e assis ência écnica nes e abalho e ao Rica do Pi es pela assis ência com o mic oscópio de o ça a ómica. E po que po ás de um abalho es á semp e uma g ande equipa, chegou a al u a ce a de ag adece a odo o g upo L-Phage. A odos eles um eno me ob igada po me e em acolhido ão bem. Que ia apenas deixa um ag adecimen o especial àqueles que acompanha am mais de pe o e que o am peças undamen ais nes a jo nada. Um ob igada à Ana B andão, ao Alex, à Cá ia, ao Da id, à Daniela, à Jonhy, à Licinha, à Lu, à Ma a, à Ri a, à Ru e e, po im, um ob igada à pessoa que ez “babysi ing” nas suas ho as agas, Ma ia, a i o meu ob igada… ocês são o que de melho le o des a expe iência. Foi um p i ilégio aze pa e des a equipa. L-Phage ocês são inc í eis. Às duas meninas que ilha am es es 5 anos lado a lado comigo, ob igada. Po meio de lág imas e so isos jun as encon amos as saídas de odos os labi in os. Ma y e B u, ocês são mui o impo an es pa a mim… que con inuemos a b ilha jun as, semp e! Pa a aqueles que ib am com as minhas conquis as como se ossem as deles, pa a aqueles que mesmo es ando a quilóme os de dis ância, nunca se esquecem de mim; pa a aqueles que semp e me apoiam incondicionalmen e… No osso olha ejo o o gulho que sen em po mim, e po isso um ob igada nunca se á su icien e. Simplesmen e, sou uma a o unada po pe ence à amília que pe enço. O ag adecimen o mais signi ican e de odos, ai pa a aqueles que me pe mi em odos os dias oa em di e en es di eções. Mãe e Pai, ob igada! Ob igada po me deixa em se a somb a mais boni a do osso e lexo. Te mino com o ag adecimen o àquele que i e na con adição comigo, mas que apesa de não demons a sei que o ce po mim… amo de i mãos é isso mesmo. “The oo s o wo k may be bi e , bu he ui is swee ” by o une cookie, 2022 i STATEMENT OF INTEGRITY I he eby decla e ha ing conduc ed his academic wo k wi h in eg i y. I con i m ha I ha e no used plagia ism o any o m o undue use o in o ma ion o alsi ica ion o esul s along he p ocess leading o i s elabo a ion. I u he decla e ha I ha e ully acknowledge he Code o E hical Conduc o Uni e si y o Minho. Cons ução De Bac e ió agos Especí icos Pa a O Diagnós ico de α-Sinucleinopa ias RESUMO As α-sinucleinopa ias são doenças neu odegene a i as ca ac e izadas po depósi os ano mais de ag egados de α-sinucleína em neu ónios ou células gliais, capazes de p o oca demência e pe u bações do mo imen o, especialmen e em pessoas idosas. De aco do com a o dem de p e alência, as ês p incipais α-sinucleinopa ias são a doença de Pa kinson, demência com co pos de Lewy e a o ia de múl iplos sis emas. A acumulação de α-sinucleína nos neu ónios, esul a nos chamados co pos de Lewy, que são a ma ca pa ológica da doença de Pa kinson e demência com co pos de Lewy, enquan o a acumulação de α-sinucleína nas células oligodend ogliais dá o igem às chamadas inclusões ci oplasmá icas gliais, que são a p incipal ca ac e ís ica da a o ia de múl iplos sis emas. A ualmen e, es as α-sinucleinopa ias não êm qualque ipo de a amen o ou diagnós ico p ecoce, pelo que o desen ol imen o de abo dagens pa a diagnós ico e e apêu ica são c uciais. Uma ez que a ag egação de α-sinucleína é uma ca ac e ís ica de odas es as doenças neu odegene a i as, as es a égias que enham es e bioma cado como al o podem o na -se e apias p omisso as. No en an o, o desen ol imen o de no as es a égias de a amen o e/ou diagnós ico é limi ado pela ba ei a hema oence álica que e es e o sis ema ne oso cen al. Pos o is o, a p esen e p opos a e e como p incipal obje i o desen ol e um sis ema baseado em bac e ió agos ( í us de bac é ias) capazes de a a essa a ba ei a hema oence álica pa a diagnos ica α–sinucleinopa ias, p ecocemen e. Nes e abalho, bac e ió agos ilamen osos M13, o am manipulados gene icamen e pa a aze em o display à supe ície de pép idos (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA e 4554WN6A: KDGIVAGVKA) po encialmen e capazes de econhece em e se liga em à α-sinucleína. Os agos de in e esse o am ob idos com sucesso po in e médio da combinação do DNA de um agemídeo modi icado com o DNA do ago aulixia M13KO7. De modo a a alia a a inidade dos agos a i iciais com a egião 45-54 da α-sinucleína e a ca ac e iza mo ologicamen e os agos aquando des a in e ação, ensaios de ca ac e ização o am ealizados. Os esul ados ob idos apenas e idencia am que os agos exibiam o espe i o pép ido de in e esse, e que a ní el ísico e químico ap esen a am um amanho e uma ca ga condizen e com o que e a espe ado. Resumidamen e, pode dize -se que os agos o am ob idos com sucesso, o que ab e po as a no as linhas de es udo u u as. Es a e amen a se á um pon o de pa ida pa a o diagnós ico p ecoce e pa a, possi elmen e, um a amen o capaz de a a a p og essão des as doenças. Pala as-cha e: α-sinucleína, α-sinucleinopa ias, bac e ió agos. i Enginee ing o speci ic bac e iophages o α-synucleinopa hies diagnosis ABSTRACT α-synucleinopa hies a e neu odegene a i e diseases ha p ima ily a ec aging people. These diseases a e cha ac e ized by abno mal deposi s o α-synuclein agg ega es in neu ons o glial cells, which cause demen ia and mo emen diso de s. In line wi h he o de o p e alence, he h ee main α- synucleinopa hies a e Pa kinson's disease, demen ia wi h Lewy bodies, and mul iple sys em a ophy. The accumula ion o α-synuclein in neu ons esul s in he so-called Lewy bodies, he hallma k o Pa kinson's disease and demen ia wi h Lewy bodies. In con as , he buildup o α-synuclein in oligodend oglial cells gi es ise o he so-called glial cy oplasmic inclusions, which a e he de ining cha ac e is ic o mul iple sys em a ophy. Cu en ly, hese α-synucleinopa hies a e no ea ly diagnosed and do no ha e any ea men , so he de elopmen o diagnos ic and he apeu ic app oaches is c ucial. Since α-synuclein is a pa hological hallma k o all hese neu odegene a i e diseases, he de elopmen o s a egies wi h his bioma ke as a a ge can be powe ul he apies. Howe e , he de elopmen o new ea men and/o diagnos ic app oaches is limi ed by he blood-b ain ba ie ha shel e s he cen al ne ous sys em. The e o e, he p esen p oposal aimed o de elop a sys em based on bac e iophages (bac e ial i uses) able o c oss he blood-b ain ba ie o ea ly diagnose α-synucleinopa hies. In his wo k, ilamen ous bac e iophages M13 we e gene ically manipula ed o display a he su ace pep ides (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA, and 4554WN6A: KDGIVAGVKA) po en ially capable o ecognizing and binding o α-synuclein. The phages o in e es we e success ully ob ained by combining DNA om a modi ied phagemid wi h DNA om he helpe phage M13KO7. In o de o e alua e he a ini y o he enginee ed phages o he 45-54 egion o α-synuclein and o mo phologically cha ac e ize he phages a he ime o his in e ac ion, cha ac e iza ion assays we e pe o med. The esul s ob ained showed ha he phages exhibi ed he espec i e pep ide o in e es , and ha a he physicochemical le el p esen ed a size and cha ge consis en wi h wha was expec ed. In summa y, i can be said ha he phages we e success ully ob ained, which opens he doo o new u u e lines o s udy. This ool will be a s epping-s one o ea ly diagnosis and possibly a ea men capable o hal ing he p og ession o hese diseases. Keywo ds: α-synuclein, α-synucleinopa hies, bac e iophages. ii INDEX RESUMO ........................................................................................................................................... ABSTRACT ....................................................................................................................................... i LIST OF ILLUSTRATIONS ................................................................................................................ x LIST OF TABLES ............................................................................................................................ xiii LIST OF ABBREVIATIONS AND ACRONYMS .............................................................................. xi 1. INTRODUCTION .................................................................................................................... 18 1.1. α-Synucleinopa hies ..................................................................................................... 18 a) Pa kinson´s disease .................................................................................................. 18 b) Demen ia wi h Lewy bodies ........................................................................................ 19 c) Mul iple Sys em A ophy ............................................................................................ 20 1.2. α-Synuclein .................................................................................................................... 21 1.3. Blood-B ain Ba ie ....................................................................................................... 23 1.4. Bac e iophages ............................................................................................................. 24 1.4.1. Phage Display echnology .................................................................................... 25 a) M13 Phage Gene ic Manipula ion .................................................................................. 29 b) M13 Phage Chemical Manipula ion ................................................................................ 29 c) M13 Phage Applica ions ................................................................................................ 30 1.5. Pep ide Ta ge ing ......................................................................................................... 32 2. MOTIVATION AND OBJECTIVES ......................................................................................... 35 3. MATERIAL AND METHODS .................................................................................................. 36 3.1. De elopmen o phagemids ......................................................................................... 36 3.1.1. Inse syn hesis ...................................................................................................... 36 3.1.2. Cloning p ocess o he inse s in o he phagemid ............................................ 38 a) Diges ion Reac ion ......................................................................................................... 38 b) Liga ion ......................................................................................................................... 39 xi LIST OF ABBREVIATIONS AND ACRONYMS A Aβ: Amyloid-β APS: Ammonium pe sul a e AS: α-Synuclein B BBB: Blood-b ain ba ie C CaCl2: Calcium chlo ide CEA: Ca cinoemb yonic an igen CECs: Ce eb al endo helial cells CED: Con ec ion-enhanced deli e y CNS: Cen al ne ous sys em CRC: Colo ec al cance D DLB: Demen ia wi h Lewy bodies DLS: Dynamic ligh sca e ing DMPS: 1,2-dimy is oyl-sn-glyce ol-3-phospho-L-se ine DNA: Deoxy ibonucleic acid DOX: Doxo ubicin E E. coli : Esche ichia coli EDTA: E hylenediamine e aace ic acid F FA: Folic acid x FDG-PET: F- luo odeoxyglucose posi on emission omog aphy F phage: Filamen ous phage FR: Fola e ecep o H HCl: Hyd ogen chlo ide G GCIs: Glial cy oplasmic inclusions GM-CSF: G anulocy e-mac ophage colony-s imula ing ac o I IG egion: In e genic egion IPTG: Isop opyl β-D-1- hiogalac opy anoside IEP: Isoelec ic poin L LB: Lu ia-Be ani medium LBs: Lewy bodies M MgCl2: Magnesium chlo ide MRI: Magne ic esonance imaging MSA: Mul iple sys em a ophy N NAC: Non-amyloid-componen NaCl: Sodium chlo ide P PCA: P o ein- agmen complemen a ion assay x i PCR: Polyme ase chain eac ion PD: Pa kinson’s disease PBS: Phospha e bu e ed saline PDT: Pho odynamic he apy PEG: Poly(e hylene glycol) PET: Posi on emission omog aphy p-S129: Se ine esidue-129 R RNA: Ribonucleic acid S SBP: SPARC-binding pep ide scF : Single-chain a iable agmen s SDS-PAGE: Sodium dodecyl sul a e- polyac ylamide gel elec opho esis SEM: S anda d e o o he mean SNARE: Soluble N-e hylmale-imide-sensi i e ac o -a achmen p o ein ecep o s SNCA: Synuclein-alpha gene SOC: Supe op imal b o h wi h ca aboli e ep ession SPARC: Sec e ed p o ein, acidic, and ich in cys eine SPECT: Single-pho on emission compu ed omog aphy ssDNA: Single-s anded DNA STEM: Scanning T ansmission Elec on Mic oscopy T TBS: T is-bu e ed saline TE: T is-EDTA TEM: T ansmission elec on mic oscopy TEMED: Te ame hyle hylenediamine TGS: T is- Glycine- SDS x ii W WT: Wild ype Z ZnPc: Zinc ph halocyanine Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 18 1. INTRODUCTION 1.1. α-Synucleinopa hies In he las wo decades α–synuclein (AS) has come in o special ocus. I is conside ed he p o ein esponsible o igge ing ce ain pa hologies ha cause demen ia and mo emen diso de s, which ha e a high incidence in he popula ion o e he age o 50-65 yea s old. Thus, in o de o p e alence, Pa kinson’s disease (PD), demen ia wi h Lewy bodies (DLB), and mul iple sys em a ophy (MSA) a e neu odegene a i e diseases ha belong o he amily o α-synucleinopa hies since hey a e cha ac e ized by abno mal deposi s o AS agg ega es in neu ons o glial cells [1] – [5]. AS agg ega ion in he neu ons esul s in abno mal deposi s called Lewy bodies (LBs). LBs a e p esen in many egions o he b ain (neoco ex, o eb ain, b ains em and o he pa s o he ne ous sys em essen ially in subs an ia nig a pa s compac a ), leading o chemical modi ica ions ha migh induce p oblems wi h hinking, beha io , mood and mo emen . LBs a e sphe ical (5-25 µm in diame e ) in aneu al cy oplasmic inclusions, mos ly consis ing o AS p o ein agg ega es su ounding hei dense nucleus. Mo eo e , hey a e a pa hological hallma k o PD and DLB [3], [6] – [8]. None heless, hey can be dis inguished by he LBs dis ibu ion. LBs a e localized in he mid-b ain, mo e p ecisely in he subs an ia nig a in PD, and in ce eb al co ex a ea in DLB [9], [10]. Howe e , he accumula ion o AS in oligodend oglial cells, p esen in he cen al ne ous sys em (CNS), is known as glial cy oplasmic inclusions (GCIs). These inclusions, which a e a gy ophilic ib illa y s uc u es composed o mis olded α-synuclein, a e he mos dis inguishing ea u e o MSA [3], [11], [12]. a) Pa kinson´s disease Pa kinson's disease (PD) is he second mos common neu odegene a i e disease a e Alzheime ´s disease, a ec ing mo e han 6 million people, being his numbe expec ed o double by 2050 [13], [14]. S a is ical da a show ha since 1990, PD cases ha e almos ipled, as om 2.5 million people diagnosed wi h PD o a o al o 6.1 million people in 2016. In 2019, mo e han 8.5 million indi iduals wo ldwide we e li ing wi h PD, ep esen ing an e en la ge popula ion han he p e iously epo ed cases o PD in 2016 [15], [16]. This α-synucleinopa hy is cha ac e ized by a massi e loss o dopamine gic neu ons in he subs an ia nig a , which leads o he appea ance o he ou classic mo o symp oms o his disease: s i ness, gai dis u bance wi h pos u al ins abili y, es emo and b adykinesia Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 19 (di icul y in ini ia ing mo emen s, which in u n a e slow). The main isk ac o o PD is age, bu en i onmen al ac o s can also be associa ed. In e ms o gende , his disease has a 1.5–2 imes highe p e alence and incidence in men han in women. In addi ion, women end o demons a e mild symp oms wi h a slowe p og ession o mo o diso de s bu ha e a highe a e o emo , abou 67% s 48% in men [17]–[21]. Nowadays, only 4% o diagnoses a e made in pa ien s unde 50 yea s o age [14]. The diagnosis o Pa kinson's disease is made on he basis o clinical symp oms. Ne e heless, o ule ou o he condi ions, magne ic esonance imaging (MRI) is o en used o e alua e he ana omy and s uc u al pa hology o he b ain so ha pa e ns o s uc u al deg ada ion can be ound o induce a co ec diagnosis. In addi ion o MRI, o he imaging s udies such as single-pho on emission compu ed omog aphy (SPECT) can be pe o med. This echnique is use ul o assessing he in eg i y o he nig os ia al dopamine gic pa hways, which in cases o PD a e ound o be dys unc ional. Mo eo e , labo a o y es s can also help diagnose PD, al hough he e a e s ill no speci ic es s o his disease [20], [22]. Cu en ly, no neu o he apy can ea , slow down and/o p e en his ch onic disease. Al hough he e is s ill no cu e o Pa kinson's disease, he e a e some d ugs o alle ia e some o he symp oms. The d ugs a e used o inc ease he le el o dopamine in he b ain (e.g. Le odopa), a ec o he neu onal chemicals (e.g. En acapone), o help con ol emo symp oms (e.g. Benzo opine) [20]. b) Demen ia wi h Lewy bodies Wo ldwide, many cases o demen ia a e diagnosed e e y day. Demen ia is de ined as a loss o cogni i e abili y ha nega i ely impac s he no mal unc ions o people's daily ou ines. The e a e many kinds o demen ia, namely demen ia wi h Lewy bodies (DLB), a p og essi e disease, which means ha symp oms s a slowly and wo sen o e ime. A e Alzheime ´s disease, i is he second mos common o m o demen ia accoun ing o 20% o demen ia wo ldwide, mainly in he age g oup o 65 yea s and olde , wi h males being mo e a ec ed [2], [23]–[27]. A he symp oma ic le el, DLB causes dis u bances in a en ion, mo emen , isuospa ial skills, and memo y ecall. Pa ien s wi h DLB commonly p esen psychia ic and beha io al dys unc ions such as sleep dis u bances, hallucina ions, and apa hy. In cases o DLB, cogni i e symp oms p ecede mo o symp oms [20], [23]. Diagnosing DLB has been a challenge, bu his is essen ially done by conside ing he symp om cha ac e is ics. Symp oma ic analysis can be supplemen ed wi h MRI o assess whe he o no he e is Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 20 b ain a ophy, o i can be u he aided by o he imaging s udies such as posi on emission omog aphy (PET) and SPECT. PET is used o assess biochemical p ocesses; hus, bo h PET and SPECT a e used o e alua e he in eg i y o he nig os ia al dopamine gic pa hways o check whe he o no dopamine anspo e up ake is being educed in he b ain. Since in DLB i has been iden i ied ha occipi al hypome abolism exis s, PET can also be used o assess possible b ain me abolism impai men s using F- luo odeoxyglucose (FDG), a adiopha maceu ical speci ic o glucose anspo and me abolism. To da e, he e a e s ill no labo a o y es s ha help diagnose DLB because no pa icula ma ke has ye been ound o his disease [20], [24], [26]–[28]. Some medica ions, such as Le odopa a e used o elie e he symp oms o DLB, bu he e is s ill no ea men ha can s op disease p og ession [3], [20]. E en hough his disease is he second mos common o m o neu odegene a i e demen ia, i is o en misdiagnosed, unde - esea ched, unde - ecognized, and unde ea ed [29]. c) Mul iple Sys em A ophy Mul iple sys em a ophy (MSA) is a a e, poo ly cha ac e ized, a al neu odegene a i e disease wi h an incidence a e o 3 cases pe 100,000 pe son-yea s o e he age o 50 [20]. This α - synucleinopa hy is highly p og essi e and a ec s mul iple neu ological sys ems including cogni i e, au onomic, ce ebella and mo o sys ems. Thus, his disease p esen s a wide ange o symp oms, including espi a o y ailu e, u ogeni al diso de , u ina y incon inence, cons ipa ion, e ec ile dys unc ion, o hos a ic hypo ension, espi a o y s ido , swea gland dys unc ion, and mo o p oblems simila o PD [3], [20], [30]–[32]. As wi h he o he wo α-synucleinopa hies, he diagnosis is made using symp oma ic es s. The use o MRI is help ul o see whe he o no ce ain a eas o he b ain ha e cha ac e is ic a ophies o MSA. Pa ien s wi h MSA a e shown o ha e egional hypome abolism in he s ia um , b ains em, and ce ebellum, so pe o ming an FDG-PET may help in he diagnosis. In con as o DLB and PD, he use o p esynap ic dopamine PET o SPECT imaging is no e y help ul. The use o labo a o y es s o con i m his disease does no ye exis [20]. Despi e he exis ence o some ea men s o alle ia e bo he some symp oms, no e ec i e he apy o MSA exis s because he mechanism o disease p og ession is unknown [3], [20]. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 21 O he h ee α-synucleinopa hies men ioned, he i s wo a e he mos p oblema ic, gi en hei s a is ical alues. Howe e , o none o he h ee, he e is an ea ly diagnosis o any ype o ea men ha allows he cu e o p e en s disease p og ession. In his way, gi en ha AS agg ega ion is a pa hological hallma k o all hese neu odegene a i e diseases, he ocus on de eloping s a egies ha a ge his p o ein agg ega es may ha e a p omising u u e as po en ial diagnos ic/ he apeu ic app oach. 1.2. α-Synuclein α-synuclein (AS) is a p o ein ha belongs o a small amily o he h ee synuclein p o eins: α- synuclein, β-synuclein and γ-synuclein. I is composed o 140 amino acids encoded by he synuclein alpha gene (SNCA) localized on ch omosome 4q21-q23, and p esen s a high solubili y, which allows i o be easily inco po a ed in o cell memb anes. I is cha ac e ized as a na i ely un olded s uc u e and sc eens wo di e en s uc u al con o ma ions: a diso de ed monome o an α-helical mul ime ic shape (Figu e 1). The monome s may i s associa e in o oligome ic species ( oxic o ms) ha subsequen ly p og ess o ib illa agg ega ed o ms (wi h β-shee con o ma ion), culmina ing in ib illa amyloid o ms, which a e p esen in Lewy bodies. Thus, he mis olding and consequen ly agg ega ion o he monome s is a key ac o in he o ma ion and p og ession o he neu odegene a i e diseases known as α- synucleinopa hies [21], [33]. Figu e 1. The α-synuclein agg ega ion p ocess (adap ed om [21]). C ea ed in BioRende .com. In addi ion, his p o ein con ains h ee dis inc s uc u al domains (Figu e 2A). The N- e minal domain ( esidues 1–60), which is o e all posi i ely cha ged, has a high con en o hyd ophobic amino acids. I is an amphipa hic lysine- ich egion cha ac e ized by epe i ions o he six lysine- ich highly Memb ane-bound helically olded monome Un olded monome ↔ Oligome Fib ils Lewy body Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 22 conse ed mo i s KTK(E/Q)GV, ha play an impo an ole in lipid binding and α-helix o ma ion. This in e ac ion o AS wi h memb ane lipids is becoming an inc easingly a ac i e a ea o unde s anding he mechanisms o AS agg ega ion, as lipids a e being iden i ied as elemen s wi h a high p eponde ance in enhancing AS agg ega ion. The AS-lipid in e ac ion can lead o he p ima y nuclea ion, which leads o he o ma ion o oxic o ms o AS - he oligome s, which consequen ly lead o he o ma ion o ib illa AS (Figu e 2B) [13], [34]–[36]. Mo eo e , ha egion, essen ial be ween esidues 45-54, has pa icula impo ance since i is in his domain whe e a e localized all known clinical mu a ions ha a e associa ed wi h he cases o DLB and PD; he e o e, hese mu a ions a e an impo an elemen o p omo e and inc ease agg ega ion o α-synuclein. The cen al hyd ophobic egion ha is ich in hyd ophobic mo i s comp ises he non-amyloid-componen (NAC) ( esidues 61–95) and esidues 71–82 (VTGVTAVAQKTV), c ucial o AS agg ega ion (mainly i s ib illa ion). Las ly, he C- e minal egion ( esidues 96–140), a nega i ely cha ged egion, is highly acidic and p oline en iched and is in ol ed in he p o ein’s chape one- like ac i i y [8], [21], [37]–[40]. Figu e 2. α-synuclein s uc u e and agg ega ion p ocess. [A] Na i e sequence o α-synuclein: clinical mu a ions (Ala30P o, Glu46Lys, His50Gln, Gly51Asp, Ala53Th , Ala53Val, and Ala53Glu) ma ked in highligh ed o ange; he amphipa hic N- e minal egion con ains six impe ec lysine- ich highly conse ed mo i epea s, ma ked in pu ple; he cen al hyd ophobic egion is unde lined and he hyd ophobic egion is ma ked in yellow; he wo majo phospho yla ion si es (Se 87 and Se 129) a e colou ed in pink, he chape one-media ed au ophagy ecogni ion si es a e ma ked in da k blue, and ni a ion si es (Ty 39, Ty 125, Ty 133, and Ty 136) a e shown in ligh blue (adap ed om [21]); [B] The α-synuclein agg ega ion media ed by he in e ac ion α-synuclein-lipid [34]. AS is p edominan ly loca ed a he p esynap ic e minals o neu ons, and al hough i s unc ion in he body is no ye ully unde s ood, i is belie ed o be in ol ed in egula ing synap ic esicle a icking in he heal hy b ain ia di ec in e ac ion wi h lipid memb anes. This unc ion is essen ial o assembling he N-e hylmaleimide-sensi i e ac o a achmen p o ein ecep o (SNARE) complexes because hese a e c ucial o eleasing neu o ansmi e s a p esynap ic ne e e minals, o ins ance, dopamine (which plays a ele an ole in mo i a ion, a en ion, egula ing body mo emen s, memo y, and cogni i e [A] M1DVFMKGLSKAKEGVVAAAEKTKQGVAEAA30GKTKEG VLYVGSKTK45EGVVHGV52AT54V55AEKTK60EQVTNV GGAVV71TGVTAVAQKTV82EGAGSIAAATGFV95KKDQLG KNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPE A140 [B] Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 23 unc ions). This highligh s he ac ha AS is a p eponde an ac o in neu odegene a ion [8], [21], [41], [38]. The mis olding and agg ega ion in i o o AS is condi ioned by se e al condi ions such as inc eased empe a u e; lipids wi h high solubili y in aqueous solu ion and sho hyd oca bon chains; molecula c owding; acidic pH; hepa in; di alen and i alen me al ions such as aluminum, coppe (II), i on (III), cobal (III) and manganese (II); o ganic sol en s; and o he glycosaminoglycans, polyca ions, pes icides and α-synuclein binding p o eins. Fu he mo e, missense mu a ions (changing a nucleo ide gi es ise o a codon ha encodes a di e en amino acid) and pos - ansla ional modi ica ions o AS, like phospho yla ion, ubiqui ina ion, ni a ion, sumoyla ion, unca ion, and N– e minal ace yla ion, a e b oadly implica ed in he p ocess o α-synuclein agg ega ion and neu o oxici y. This occu s because he accumula ion o he AS agg ega es in p esynap ic e minals o he axons u n he cells mo e sensi i e o a a ie y o oxic inju ies [8], [38], [42]. The mos o dina y pos - ansla ional modi ica ion is phospho yla ion, as abou 90% o he AS agg ega es in Lewy bodies a e phospho yla ed on he se ine esidue-129 (p-S129). The ubiqui ina ion in lysine esidues (mainly a K6, K10, and K12 esidues) and he ni a ion in y osine (Y39, Y125, Y133, and Y136), a e he o he wo mos common pos - ansla ional modi ica ions [8], [38]. In his way, i is no o ious ha AS has a undamen al ole in he de elopmen o α- synucleinopa hies. 1.3. Blood-B ain Ba ie He eupon, i is c ucial o c ea e s a egies o ea men and/o diagnosis o α-synucleinopa hies. Howe e , he anspo o mos he apeu ic molecules o he b ain is blocked by he exis ence o a physical, me abolic, anspo and immunological ba ie , which main ains homeos asis be ween he blood and he b ain. This na u al ba ie , which p o ec s he cen al ne ous sys em (CNS), is called blood- b ain ba ie (BBB) and ep esen s a signi ican challenge o de eloping app oaches o ea men and/o diagnosis o neu ological diseases. BBB is a highly selec i e semipe meable ba ie ha con ols he in lux and e lux o di e en subs ances. This ba ie only enables a speci ic exchange o nu ien s, ions, me abolic was e p oduc s, mine als and signaling molecules be ween he blood and he b ain. I also has he aim o p o ec ing he b ain om physiological luc ua ions o he plasma and om blood-bo ne compounds ha may in e pose wi h he no mal neu o ansmission p ocess. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 30 This in oduc ion o unc ional de i a i es in he coa p o eins allows su ace modi ica ion o he phages wi h a ious chemicals such as ch omopho es, luo escen dyes, enzymes, and syn he ic oligome s, playing an essen ial ole in a panoply o applica ions such as bioimaging, issue enginee ing and biosenso s [67]. c) M13 Phage Applica ions Biosa e y, mass manu ac u ing wi h uni o m pa ame e s a a low cos , and all he in o ma ion p esen ed he e show why his phage o e s po en ial o nume ous applica ions in di e en a eas [80]. As al eady men ioned, phages and Phage Display ha e been used o clinical usage. Mu gas e al . demons a ed ha M13 phages ha e a high po en ial as an immuno he apeu ic s a egy o colo ec al cance (CRC). This cance is qui e le hal, and cu en ly, he e a e no e ec i e con en ional ea men s. Thus, since his pa hology is cha ac e ized by high exp ession o he ca cinoemb yonic an igen (CEA) ha p omo es he agg a a ion o his umo 's magni ude, his was he main a ge . This g oup o esea che s, h ough a sys emic adminis a ion o CEA-speci ic M13 phages (M13 phages ha exhibi ed in he p3 p o ein o i s capsid, single-chain a iable agmen s (scF ) de i ed om a monoclonal an ibody speci ic o CEA), in mice wi h CRC showed a ma ked educ ion in he g ow h o his umo [81]. Mo e ecen ly, Han e al . designed a new pu pose agains his umo al ac i i y, whe ein he majo coa p o ein, p8, o he M13 phage displayed an an i umo cy okine, g anulocy e-mac ophage colony-s imula ing ac o (GM- CSF), which signi ican ly dec eases umo g ow h in mu ine. Fu he mo e, his app oach combined wi h adia ion imp o ed he pe o mance o his he apy [82]. In he cu en days, wi h he con inuous inc ease o d ugs esis ance, phage he apy can be a new solu ion, as Lu & Collins showed. Thei s udy demons a ed ha ans e ing genes ha dec ease he SOS esponse o bac e ia o he dys unc ions caused by an ibio ics ia he M13 phage weakened he bac e ia making hem mo e suscep ible o an ibio ics [83]. Filamen ous phages can also be used as biosenso s, which is help ul o isualizing he p og ession o diseases in such a way ha ele an in o ma ion can be ob ained o diagnosis and ea men . This is possible, as he phage capsid con ains unc ional g oups wi h a ying eac i i y, whe e mul iple syn he ic molecules can be selec i ely bound o he phage sca old. These phages a e mainly p esen ed as a nanop obe by conjuga ing luo escen molecules and a ge ing moie ies on he capsid su ace. Li e al . o example, enginee ed a dual-modi ied M13 pa icles able o de ec human KB umo cells by emi ing luo escence upon binding o olic acid (FA) o he umo cells. The FA is a i amin ha plays an impo an ole a he cellula le el, and i is aken up by he ola e ecep o (FR) p esen in cells Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 31 and o e exp essed in cance . The dual-modi ica ion on he phage consis ed o chemical modi ica ions o he subuni s o he p8 p o ein, which allowed he binding o he olic acid and luo escen molecules [84], [85]. Gosh e al . de eloped M13 phages as a magne ic nanopa icle ehicle o in i o p os a e cance imaging. In hei wo k, he M13 phage genome was modi ied so ha he mino capsid p o ein, p3, exhibi ed a a ge ing pep ide wi h a ini y o SPARC (Sec e ed, Acidic, Cys eine Rich P o ein), a glycop o ein highly exp essed in melanoma, agg essi e melanoma, b eas , b ain, p os a e, colon, and lung cance . The pep ide used is known as SPARC-binding pep ide (SBP). Conce ning he p8 p o ein, i has been chemically al e ed o house magne ic i on oxide nanopa icles (MNPs). Nanopa icles a e p omising con as agen s o dynamic molecula imaging due o hei in insic op ical, elec ical and magne ic p ope ies, so MNPs we e used o de ec speci ic umo s ia da k con as magne ic esonance imaging. The esul s o his s a egy sugges ed ha he use o M13 compa ed o nanopa icles, ha we e di ec ly unc ionalized wi h a ge pep ides, was mo e e ec i e o ampli ied a ge ing in i o and was able o ob ain be e da k con as imaging in i o , as each SPARC a ge molecule deli e ed a la ge numbe o nanopa icles o he cells [86]. Mo eo e , Gosh e al ., in 2012, used he M13 phage as a p omising pla o m o p os a e umo cell imaging and d ug deli e y. They gene ically manipula ed he M13 phage so ha he p3 p o ein was used o SPB. In addi ion, he phage was also gene ically and chemically unc ionalized so ha he d ug doxo ubicin (DOX), commonly used in chemo he apeu ic sec ions, was coupled o he p8 p o ein subuni s, and also so ha luo escen molecules (s ep a idin Alexa Fluo ® 488 Alexa) we e p esen ed on he su ace o he p9 p o ein o de ec he p os a e cance cells. This whole phage complex has been demons a ed in i o o be a po en ial moni o ing ool due o he acqui ed luo escence. Besides ha , his phage app oach was p o ed o ha e he apeu ic po en ial as i was able o dec ease no only he iabili y o he cance cells bu also educed he concen a ion o he applied d ug, as i is a a ge ed d ug deli e y [87], [88]. Liposomes a e a i icial phospholipid esicles cha ac e ized by easy deg ada ion, li le o no side e ec s, high loading capaci y and good biocompa ibili y, making hem sui able d ug ca ie s. Howe e , his ype o d ug deli e y is no s able in he biological sys em, so Ngweni o m e al . de eloped a phage M13-liposome complex o op imize he pe o mance o hese nanomolecules. Th ough elec os a ic in e ac ions, igge ed by he nega i ely cha ged pep ide displayed on he majo i y p o ein o he p o ein coa , he liposomes we e coupled o he phage. Inside, he liposomes we e loaded wi h zinc ph halocyanine (ZnPc), a d ug es ed as a pho osensi ize o pho odynamic he apy (PDT). This Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 32 nanos uc u e has been shown o ha e he po en ial o be used o a ge ed d ug deli e y in PDT and o eaching speci ic cells [89], [90]. Those wo ks showed di e en examples whe e he M13 ilamen ous phage is used as a pla o m. Thus, his phage can be applied as an inno a i e s a egy o diagnose/ ea α-synucleinopa hies. Being his pu pose powe ed by he wo ks o Hemi Diman and hei colleague, ha desc ibed he na u al capaci y o phage M13 o disagg ega e α-synuclein agg ega es in i o . They obse ed ha when his phage is incuba ed wi h an α-synuclein ib il solu ion, ib il disagg ega ion occu ed h ough a possible in e ac ion o he ilamen ous phages o a hyd ophobic s e ch in he cen al hyd ophobic egion spanning amino acids 73–85 [37], [91]–[93]. This abili y is an exci ing and ele an skill o his wo k. 1.5. Pep ide Ta ge ing Di ec ly a ge ing he agg ega ion p ocess is an appealing he apeu ic s a egy. Hence, he in e es in using pep ides in his a ea, especially when i comes o neu odegene a i e esea ch, has been g owing. This happens because pep ides ha e a high po en ial o p e en ib illa o ma ion, as hey a e able o block he su ace p o ein-p o ein in e ac ions o med du ing he agg ega ion p ocess, which is no achie ed by mos o he small molecules ha cons i u e he comme cial d ugs. Mo eo e , pep ides ha e a highe speci ici y o a ge ing, which leads o a educ ion in undesi able side e ec s [94]. In 2015, Che u a a e al . designed a mul iplexed in acellula p o ein- agmen complemen a ion assay (PCA) lib a y sc eening sys em based on he na i e AS sequence 45-54, which is desc ibed as he egion o AS ha con ains missense mu a ions implica ed in he agg ega ion p ocess. Using his sequence as a empla e, hey c ea ed a 209,952-membe pep ide lib a y wi h en amino acids in leng h ha spans esidues 45–54 (KEGVVHGVAT). F om his lib a y, he pep ide 4554W (K1DGIVN6GVKA) has been iden i ied as a po en ial candida e ha showed in in i o assays educed AS ib il o ma ion and cy o oxici y [95]. Recen ly, To pey and hei colleagues, based on he wo k o Che u a a e al ., epo ed ha 4554W pep ide can bind o pa ially o wholly agg ega ed AS species, p e en ing u he ib illa ion and disagg ega ing p e- o med AS ib ils esul ing in sho e ib ils. Addi ionally, hey did no obse e any associa ion o he pep ide wi h monome ic AS, which means ha he 4554W pep ide ((KDGIVNGVKA)) does no in e e e wi h he na i e unc ion o AS [94]. Then, Meade e al . s udied he ole o he in e ac ion be ween AS-lipid in he p ima y nuclea ion since p ima y nuclea ion is he i s s ep o agg ega ion. Thei Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 33 expe imen used 1,2-dimy is oyl-sn-glyce ol-3-phospho-L-se ine (DMPS) esicles as a phospholipid model, since hey a e an impo an elemen in dopamine gic synap ic esicles, which lead o AS memb ane binding, and consequen ly an inc ease o local concen a ion o AS ha accele a es p ima y nuclea ion. The esul s indica ed ha 4554W inhibi s lipid-induced p ima y nuclea ion o AS in i o . The e o e, he 4554W has a c ucial ole in he p ima y nuclea ion, which is impo an because he p ima y nuclea ion is conside ed a c i ical s ep o he neu o oxici y associa ed wi h AS and a desi ed poin o inhibi o unc ion [96]. Mo e ecen ly, Meade e al . published a wo k whe e hey op imized he pep ide 4554W. They pe o med a comple e alanine scan analysis (each esidue o 4554W pep ide was eplaced o an alanine). Indeed, hey e i ied ha he subs i u ion N6A co esponding o he pep ide 4554WN6A (K1DGIVA6GVKA) exhibi ed a signi ican inc ease in pep ide e icacy in inhibi ing lipid-induced p ima y nuclea ion, which sugges s ha he o he esidues o he o iginal pep ide ha e an impo an unc ion in he inhibi o y e ec o he agg ega ion pa hway. In ending o educe he size o he 4554WN6A pep ide o he smalles unc ional uni equi ed in he ac i i y, hey ound ha unca ion a he N- e minus o he pep ide, which esul ed in he dele ion o he K amino acid om posi ion 1, ga e ise o he 4654WN6A pep ide (DGIVA6GVKA). This new pep ide e idenced a signi ican inc ease in educing he numbe o oxic oligome ic species in lipid memb anes, blocking AS agg ega ion and associa ed cy o oxici y in i o , which means ha he K esidue is indispensable. This s udy also e idenced ha pep ides 4554W, 4554WN6A and 4654WN6A, being an agonis s o AS agg ega ion and associa ed oxici y, do no di ec ly p e en he binding o AS o lipids, which is a desi able ac o since he associa ion o AS o small synap ic neu o ansmi e esicles should no be dis up ed, so no o in e e e wi h he no mal ac i i y o he dopamine gic esicle usion and ecycling p ocess [34]. In May o his yea , Meade and colleagues used PCA me hodology o sc een he 45-54 pep ide lib a y, bu his ime agains i e o he cha ac e is ic clinical mu a ions in his 45-54 egion (A30P, E46K, H50Q, G51D, and A53T), wi h he goal o iden i ying a pep ide ha would be e ec i e no only agains he amilial AS a ian om which i was selec ed, bu also agains wild- ype (WT) AS. The sc eening iden i ied i e pep ides: A30PW (EEGVIDGIAA), E46KW (KEGVVNVVKA), H50QW (ENGVVNGVAT), G51DW (EDGVVDGVDA), and A53TW (EDAVVNGVAA), all o which had common esidues a some places bu di e ed a o he s. All educed agg ega ion o he ele an a ge , and some we e shown o educe agg ega ion when incuba ed wi h o he a ian s. The G51DW pep ide demons a ed he be e ac i i y among hese pep ides. In addi ion o he new pep ides iden i ied, he p e iously op imized pep ide, 4554WN6A, was e-pe o med in his s udy, concluding ha i is no only highly e ec i e agains WT AS Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis INTRODUCTION 34 bu also agains se e al mu an o ms. In he cell oxici y s udies, howe e , whe e 4554WN6A excelled in eco e ing oxici y, G51DW had he e e se impac and appea ed o inc ease oxici y o some AS a ian s. This highligh s he p omising na u e o pep ide 4554WN6A o diagnos ic and he apeu ic applica ions [97]. Based on all he a o emen ioned, i can be in e ed ha he cons uc ion o a phage pa icle exp essing pep ide ligands capable o iden i ying AS agg ega es pa es he way o de eloping a no el app oach o ea ly diagnosis, p e en ion, and/o ea men o α-synucleinopa hies. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MOTIVATION AND OBJECTIVES 35 2. MOTIVATION AND OBJECTIVES α-synucleinopa hies a ec millions o people wo ldwide. The de elopmen o new s a egies o diagnosis and/o ea men o hese neu odegene a i e diseases is u gen o p e en i s onse and possibly hal /inhibi disease p og ession. Achie ing his ea will be an amoun o e u ning people wi h hese diseases o no mal li es. Howe e , he p og ess o new app oaches is limi ed by he BBB ha houses he CNS, and cu en ly, he e is no e ec i e app oach de eloped o ea ly diagnose no ea α- synucleinopa hies. The e o e, he p esen p oposal aims o de elop a phage-based sys em capable o de ec ing he α-synuclein agg ega es by shu ling α-synuclein speci ic pep ides ac oss he BBB able o ecognize and bind α-synuclein agg ega es in he b ain. He eupon, his p ojec in ol es h ee main objec i es: i) he de elopmen o a phagemid con aining he DNA sequence o α-synuclein speci ic pep ides, wi h high binding po en ial o α-synuclein agg ega es; ii) he de elopmen o enginee ed phages con aining he pep ide(s) o in e es ; and iii) cha ac e iza ion assays incuba ing he enginee ed phages wi h α-synuclein o assess hei binding capaci y o he 45-54 egion o α-synuclein, and check he mo phology ha phages and p o ein adop when hey bind. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 36 3. MATERIAL AND METHODS This p ojec includes h ee main asks: syn hesis o he phagemids con aining he pep ides o in e es , enginee ed M13 phage p oduc ion, and cha ac e iza ion assays. 3.1. De elopmen o phagemids This ask in ol es de eloping h ee phagemids con aining he DNA sequence o α-synuclein speci ic pep ides (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA, and 4554WN6A: KDGIVAGVKA). The pep ides 4554W and 4554WN6A ha e been epo ed in he li e a u e o ha e high po en ial a ini y o α- synuclein. These pep ides we e gene a ed based on he sequence o he 45-54 AS egion. Howe e , hey di e om he na i e sequence. Thus, in o de o ha e a pep ide ha unc ions as a con ol in ela ion o he o he wo, he 4554 pep ide was designed in his wo k. The sequence o 4554 pep ide co esponding o he na i e egion 45-54 o AS, wi hou any modi ica ion. 3.1.1. Inse syn hesis In his wo k, h ee pep ides (4554, 4554W, 4554N6A) will be displayed on he su ace o he M13 phage, namely used o p o ein 3. The i s s ep is he syn hesis o he pep ides o in e es . This p ocess was based ollowing he Rangel me hod: annealing o wo p ime s wi h 100 % complemen a i y was pe o med, ollowed by a hea ing and cooling p o ocol, o ob ain he inse s [98]. Fo ha , i was i s necessa y o design he p ime s ( e e se and o wa d) o each pep ide. The amino acid sequences o he pep ides o in e es we e con e ed o nucleo ide sequences using he speci ic codons o each amino acid, o ob ain he p ime s. I should be no ed ha , in addi ion o he sequence o each pep ide o in e es , each p ime also includes an enzyme es ic ion si e o a pos e io cloning p ocess. The sequences o he p ime s a e ep esen ed in Table 1. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 37 Table 1. P ime s used o ob ain he sequences o he desi ed pep ides: In blue he es ic ion si e o he SacI enzyme; in g een he SalI enzyme es ic ion si e and in yellow he pep ides o in e es . P ime s Sequence 5’-3’ Base Pai s (bp) Pep ide Sequence 4554 (Fowa d) TATATAGAGCTCAAGGAGGGCGTGGTGCACGGCGTGGCCACCCGTCGACTATATA 55 KEGVVHGVAT 4554 (Re e se) TATATAGTCGACGGGTGGCCACGCCGTGCACCACGCCCTCCTTGAGCTCTATATA 4554W (Fowa d) TATATAGAGCTCAAGGACGGCATCGTGAACGGCGTGAAGGCCCGTCGACTATATA 55 KDGIVNGVKA 4554W (Re e se) TATATAGTCGACGGGCCTTCACGCCGTTCACGATGCCGTCCTTGAGCTCTATATA 4554WN6A (Fowa d) TATATAGAGCTCAAGGACGGCATCGTGGCCGGCGTGAAGGCCCGTCGACTATATA 55 KDGIVAGVKA 4554WN6A (Re e se) TATATAGTCGACGGGCCTTCACGCCGGCCACGATGCCGTCCTTGAGCTCTATATA In his eac ion, he indi idual p ime s o each desi ed inse (p oduced by STAB VIDA, Po ugal) we e able o anneal, he e o e c ea ing a double-s anded DNA p oduc con aining he inse o he speci ic sequence. All p ime s we e esuspended in nuclease- ee wa e o a inal concen a ion o 100 µM. The annealing p ocess was pe o med by adding equimola amoun s o each p ime (20 µL o each espec i e p ime a a concen a ion o 10 µM). Then, 10 µL o T is- hyd ochlo ide (HCl) a 10 mM, pH8, was added o a inal olume o 50 µL. The annealing was ca ied ou ollowing he condi ions desc ibed in Table 2, in a MyCycle TM The mal Cycle (Bio-Rad Labo a o ies, Inc.). Then, he concen a ion was de e mined using a NanoD op One Mic o olume UV-Vis Spec opho ome e (The mo Scien i ic) and he inse s we e s o ed a -20 °C un il usage. Table 2. Annealing condi ions. S ep Tempe a u e (°C) Time (min) 1 93 3 2 80 20 3 75 20 4 70 20 5 65 20 6 40 60 7 4 ∞ Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 38 3.1.2. Cloning p ocess o he inse s in o he phagemid Once he syn hesis o he inse s is comple ed, he nex ask was he liga ion be ween pep ides (inse s) and ec o (phagemid pETDue -1 p e iously cloned wi h he gene 3 o M13). This ask comp ises he s eps depic ed in Figu e 6. Figu e 6. Schema ic ep esen a ion o he diges ion: liga ion p o ocol (c ea ed wi h Snapgene so wa e). a) Diges ion Reac ion Be o e p oceeding o he cloning p ocess, i was i s necessa y o diges he inse s and he ec o wi h he es ic ion enzymes, SacI (The mo Scien i ic, Lo 00544923) and SalI (The mo Scien i ic, Lo 00433634). The ec o used was based in he comme cial plasmid pETDue -1 (Addgene, 71146). This plasmid has been p e iously manipula ed in o de o con ain pa o a phage genome, in his case, he gene 3 esponsible o codi ying he M13 phage mino coa p o ein 3 and he signal sequence peIB, which was p e iously added o p omo ing phage p o ein ansloca ion h ough he bac e ial memb ane and assembly in phage pa icles (Figu e 7). Figu e 7. Ge al scheme o he main egions o pETDue -1 phagemid. C ea ed in BioRende .com. [a] DIGESTION [b] LIGATION Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 39 The diges ion o he inse s and ec o is essen ial because i will allow he cloning p ocedu e. Mo eo e , he enzymes used a e unique cu e s ha o m complemen a y s icky ends in he ec o and inse s and consequen ly enable he inse o be in oduced immedia ely be o e gene 3. The ec o DNA used o he diges ion had been isola ed p e iously wi h he ZR BAC DNA Minip ep ki (ZYMO) ollowing he manu ac u e 's ins uc ions and was s o ed a -20ºC. The inse s and ec o DNA we e diges ed ollowing he condi ions o Table 3 (no e ha di e en condi ions we e es ed and ha he op imized condi ion was he one selec ed). Then, he samples we e incuba ed a 37 °C o 2 hou s, ollowed by a 15 minu es enzyme inac i a ion s ep a 65 °C. Table 3. Reac ion condi ions o ec o and inse diges ion. b) Liga ion Subsequen ly o he diges ion p ocess, he ollowing s ep was o clone he diges ed inse s in o he diges ed ec o . In his eac ion, he T4 DNA ligase enzyme was used (NewEngland BioLabs® Inc, M0202S). This enzyme joins he cohesi e end e minals o double-s anded DNA by ca alyzing he c ea- ion o a phosphodies e link be ween jux aposed 5' phospha e and 3' hyd oxyl e minals. The e o e, he liga ion was ca ied ou ollowing he condi ion on Table 4, using a 3:1 inse mola excess o 20-100 ng ec o a io. Fi s , he eac ion was mixed and incuba ed a 16 °C o e nigh . Then, he T4 DNA ligase was inac i a ed a 65 °C o 10 minu es. Subsequen ly, he liga ion p oduc was kep a -20 °C un il bac e ial ans o ma ion. No e ha di e en condi ions we e es ed and ha he op imized condi ion was he one selec ed. Vec o Inse DNA 1500 ng 1000 ng SacI 1 µL SalI 1 µL 10x Fas Diges bu e (The mo Scien i ic, Lo 00440534) 2 µL Nuclease ee wa e Up o 20 µL Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 46 mas e mix shown in Table 7. Then, he PCR eac ion o he sample was pe o med in MyCycle TM The mal Cycle (Bio-Rad Labo a o ies, Inc.) acco ding o he condi ions p esen in Table 6. The co ec leng h o he PCR p oduc was con i med by loading 6 µL o he PCR p oduc in a 2.5 % aga ose gel. A e gel con i ma ion, he PCR p oduc was cleaned and pu i ied using he DNA Clean & Concen a o TM - 5 ki (ZYMO) ollowing he manu ac u e 's ins uc ions, and he concen a ion o he DNA was quan i ied in NanoD op One Mic o olume UV-Vis Spec opho ome e s equipmen (The mo Scien i ic). Table 7. PCR p o ocol. Las ly, samples we e p epa ed wi h 500 ng o cleaned PCR p oduc , 3 μl o e e se p ime (#7, see ANNEX I) a a concen a ion o 10 mM, which ampli ies he egion o in e es o he gene 3, o check he p esence o pep ide sequences o in e es , and wa e o a inal olume o 10 μL was achie ed. The samples we e sequenced by Eu o ins. 3.3. Cha ac e iza ion assays Once he bac e iophages ha exhibi he pep ides o in e es we e success ully enginee ed: i) phage 4554 (phage F1) ha displays he KEGVVHGVAT pep ide sequence (co esponding o he na i e egion), ii) phage 4554W (phage F2) ha displays he KDGIVNGVKA pep ide sequence, and iii) phage 4554WN6A (phage F3) ha displays he KDGIVAGVKA pep ide sequence, all he condi ions we e me o mo e on o he s ep ha p o ided in o ma ion on he pe o mance o he de eloped phages. To his end, moni o ed cha ac e iza ion assays we e pe o med, whe e comme cial α-synuclein (1MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTK45EGVVHGVAT54VAEKTK60), (RayBio ech, Inc), wi h a molecula weigh o 6.1 kDa con aining he N- e minal amphipa hic domain (Figu e 9), was incuba ed wi h each o he phages displaying he di e en pep ides. These assays aimed o p o ide insigh s in o he in e ac ions be ween he di e en pep ides and he a ge , i.e., o in e p e hei abili y o bind o AS and whe he he pep ides ac ually in e ac wi h he 45-54 egion o AS. Consequen ly, he Componen s PCR ube 10 μM Fo wa d P ime (#41/#42/#43) 1 µL 10 μM Re e se P ime (#25) 1 µL Xpe Fas Mas e Mix (Re : QE15, G isp) 12.5 µL DNA 100 – 200 ng H2O ee nuclease Up o 25 µL Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 47 pe o mance o each pep ide exhibi ed by phage M13 was e alua ed and compa ed wi h each o he . In addi ion, hese assays we e in ended o supply in o ma ion abou he cha ac e is ics o he a i icial phages p oduced. Figu e 9. A schema ic ep esen a ion o he I-TASSER p o ocol o p o ein s uc u e [99]–[101]. 3.3.1. AS sample The AS used o he expe imen s was a comme cial p o ein esuspended in 50 µL o a solu ion o 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 o a inal concen a ion o 1 mg/mL. 3.3.2. Phage samples p epa a ion Be o e p oceeding wi h he expe imen s, i was necessa y o p epa e aliquo s o each phage a a concen a ion o 1 x 109 PFU/mL esuspended in he same bu e as ha o he p o ein (20 mM T is-HCl + 100 mM NaCl, pH= 7.5). Thus, in 2 mL eppendo ubes, 2 mL o each phage was added om i s espec i e s ock solu ion s o ed a 4 °C (Table 8). Then, he samples we e cen i uged a 1000 pm o 20 min a 4 °C and he supe na an was ho oughly disca ded. The esul an pelle om each sample was esuspended in 2 mL o bu e o a concen a ion o 1 x 109 PFU/mL. All samples we e s o ed a 4 °C un il u he use. α-Synuclein (1-60 egion) Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 48 Table 8. Phages concen a ions in s ock. 3.3.3. Dynamic ligh sca e ing (DLS) measu emen s The size dis ibu ion and he Z- po en ial o he phages (M13 (con ol), F1, F2, and F3) and he phages incuba ed wi h AS a a inal concen a ion o 6.25 µg/mL (M13+AS, F1+AS, F2+AS, and F3+AS) was measu ed by DLS, using Ze asize Nano ZS (Mal e n Ins umen s) a 25 °C. In an app op ia e cell DTS0012 o DTS1070, depending on he pa ame e ha is being measu ed, size o Z- po en ial, espec i ely, 800 µL o he p epa ed sample we e added. The sample was hen placed in he equipmen and ead in iplica e, being he esul s desc ibed as mean ± s anda d de ia ion. I is wo h no ing ha he phage samples we e es ed unde ou condi ions: no incuba ion, 5 minu es, 24- and 48-hou s incuba ion a 36.6 °C. On he o he hand, he samples con aining phage + AS we e only exposed o he las h ee condi ions. No e ha he selec ed incuba ion empe a u e was 36.6 ºC, as his is he mice's body empe a u e [102]. The es s we e simula ed wi h his empe a u e wi h a iew o possible u u e in i o es s. 3.3.4. Sodium dodecyl sul a e - polyac ylamide gel elec opho esis (SDS-PAGE) In o de o asce ain which o he enginee ed phages had a g ea e in e ac ion wi h he p o ein, he ee p o ein (which did no bind o he phage) in each sample was elu ed and un on an SDS-PAGE gel. a) P epa a ion o he samples To eco e he p o ein in solu ion, all he samples used in he p e ious p ocedu e we e eused and subjec ed o a spin down. Then he con en o each sample was ans e ed o new 1.5 mL Eppendo ubes and 100 µL o Coomassie blue dye (The moScien i ic) was added o each sample. Subsequen ly, he samples we e o exed igo ously and we e le o incuba e a oom empe a u e. A e 30 minu es o incuba ion he samples we e cen i uged o 10 minu es a 17000 pm so ha he p o eins p esen in solu ion and s ained wi h blue would p ecipi a e. The esul ing supe na an was emo ed, and he pelle was placed o d y a 39 ºC and s o ed a oom empe a u e un il u u e use. All s o ed samples we e Phage Pep ide Displayed S ock Concen a ion (PFU/mL) M13 ------------ 3.6 x 1010 F1 4554 2.2 x 1010 F2 4554W 1 x 1010 F3 4554WN6A 7 x 109 Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 49 esuspended in 20 µL o 1 x T is- Glycine- SDS (TGS) bu e (Nzy ech) and le o incuba e o 2 - 3 hou s. Then only he con en o he samples con aining phage and AS was aspi a ed and ans e ed o he espec i e Amicon® Ul a 0.5 mL column (Me ck). In o de ha he ee p o ein in solu ion was elu ed, he samples we e cen i uged o 15 minu es a 13300 pm and he supe na an was collec ed in new 1.5 mL Eppendo ubes. b) P epa a ion o he SDS-PAGE (15 % (w/ ) ac ylamide) gel Be o e beginning he gel p epa a ion, he elec opho esis sys em's glasses we e washed wi h dis illed wa e , d ied, and cleaned wi h alcohol o ensu e ha all he g ease has been emo ed. Then, a e assembling he gel suppo , he esol ing gel a 15% ( ha allows he sepa a ion o p o eins based on hei molecula weigh ) and he s acking gel ( ha aligns all he p o ein samples loaded in he gel, so ha hey can en e he esol ing gel a he same ime), we e p epa ed acco ding o he speci ica ions desc ibed in Table 9. B ie ly, he esol ing gel was i s p epa ed and pipe ed immedia ely on o he gel ca ie . This was sealed by a hin line o dis illed wa e , which was emo ed once he esol ing gel polyme ized. Following ha , he s acking gel was p epa ed and p omp ly pipe ed on op o he esol ing gel, ill he uppe limi o he glasses was eached. Finally, he comb was injec ed quickly a e pipe ing o minimize e en ion and/o bubble o ma ion, and he gel was allowed o polyme ize. I should be emphasized ha APS (Ammonium Pe sul a e) and TEMED ( e ame hyle hylenediamine) a e polyme izing agen s, so hey we e he las eagen s o be in oduced when p epa ing he gels. Table 9. SDS – PAGE. Reagen s Resol ing gel (15 %) S acking gel (6 %) H2O ee nuclease 2.8 mL 2.9 mL 40 % Ac ylamide/Bis – Ac ylamide 3 mL 0.750 mL 1.5 M T is- HCl, pH= 8.8 2 mL -------------- 0.5 M T is- HCl, pH= 6.8 -------------- 1.25 mL 10% SDS 80 µL 50 µL 20 % APS 80 µL 50 µL To al 8 mL 8 mL c) Applica ion o he samples o be loaded on o he gel The samples p esen in Table 10 we e loaded on o he gel. Howe e , i was i s necessa y o p epa e he samples o analysis, so o 8 µL o each sample, 2 µL o 5 x SDS- PAGE Sample Loading Bu e (Nzy ech) was added. Nex , 10 µL o each p epa ed sample was loaded in o he espec i e well o Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 50 he gel, as well as 5 µL o he PageRule TM Uns ained B oad Range P o ein Ladde (The moScien i ic, 01135959). The elec opho esis was ca ied ou in 1 x TGS bu e , and he powe supply was au oma ed o 30 minu es a 80 V, un il he samples pass h ough he concen a o gel and a e comple ely in he sepa a o gel. Subsequen ly, he ol age was inc eased o 120 V o 40 minu es. Table 10. Samples loaded on SDS-PAGE gel Sample Desc ip ion 1 Reco e ed p o ein om M13 phage sample 2 Elu ed p o ein om sample wi h M13 and AS 3 Reco e ed p o ein om F1 phage sample 4 Elu ed p o ein om sample wi h F1 and AS 5 Only AS P o ein (a a inal concen a ion o 6.25 µg/ mL) 6 Reco e ed p o ein om F2 phage sample 7 Elu ed p o ein om sample wi h F2 and AS 8 Reco e ed p o ein om F3 phage sample 9 Elu ed p o ein om sample wi h F3 and AS d) P o ein S aining - BlueSa e p o ein s ain: A e he elec opho esis p ocess, he gel was ca e ully emo ed om he elec opho esis sys em and washed wi h dis illed wa e . I was hen placed in he s aining solu ion, BlueSa e p o ein s ain (Nzy ech, MB15201), o e nigh . The nex day, he s aining solu ion was disca ded, and he e ealed bands we e analyzed. Finally, he gel was p ese ed in dis illed wa e . - Sil e s ain o p o ein gel: Fo he pu pose o g ea e de ec ion o banding pa e ns o med a e he elec opho esis p ocess he s aining can be done in sil e ni a e. Sil e s aining p o ides a high deg ee o pep ide co e age while main aining excellen sensi i i y (in he e y low nanog am ange). B ie ly, he s aining p ocess sequen ially consis s o p o ein ixa ion, sensi iza ion, washing, sil e imp egna ion and inally image de elopmen [103], [104].Thus, ollowing he p o ocol o sil e s aining he gel was i s insed in dis illed wa e . A e 10 minu es, he dis illed wa e was emo ed, and sodium hiosul a e a 0.2 g/L (VWR P olabo Chemicals, 16I154125) was added o he gel and le in con ac wi h he gel o 1 minu e. Then, he sodium hiosul a e solu ion was emo ed, and he gel was washed 2 x 20 seconds in dis illed wa e . The dis illed wa e was emo ed, and he gel was imp egna ed in a sil e ni a e solu ion a 2 g/L (VWR Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 51 P olabo Chemicals, 13C250013) du ing 30 minu es. Subsequen ly, he sil e ni a e was emo ed, and he gel was insed wi h dis illed wa e o 10 seconds. The eina e , he dis illed wa e was disca ded and he de elope solu ion (0.7 mL/ L o maldehyde 37- 38 % w/w s abilized wi h me hanol PA-ACS (Pan eac, 131328.1211), 30 g/ L sodium ca bona e anhyd ous (Pan eac, 4D013888) and 10 mg/ L sodium hiosulpha e) was added 2 x 3 minu es and he gel was shaken ca e ully un il he desi ed colo was achie ed. The de elope solu ion was emo ed, and he gel was incuba ed wi h he s op solu ion (50 g/L T is-Base (Fishe BioReagen sTM, 153464) and 2.5 % ace ic acid glacial (Fishe BioReagen sTM, 1733740) o 1 minu e. Finally, he p o ocol ended by washing he gel in dis illed wa e and he de eloped bands we e analyzed. 3.3.5. Scanning T ansmission Elec on Mic oscopy (STEM) The mo phological analyses o he 8 samples desc ibed in he Table 11 we e pe o med in an Ul a-high esolu ion Field Emission Gun Scanning Elec on Mic oscopy (FEG-SEM), NOVA 200 Nano SEM, FEI Company. Be o e analyses, samples we e ins alled in Cu-C g ids (AGAR S160-4), by imme sion in he solu ion con aining he phages o phages and AS. Then he samples we e analyzed wi h an accele a ion ol age o 15 kV, using a scanning ansmission elec on de ec o (STEM). The image iles we e examined and con e ed in o TIF o ma . Table 11. Samples analyzed in STEM echnique Sample Desc ip ion Condi ions M13 Sample con aining M13 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 Wi hou incuba ion F1 Sample con aining F1 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F2 Sample con aining F2 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F3 Sample con aining F3 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 M13 + AS Sample con aining M13 a 1 x 109 PFU/mL and AS a 6.25 µg/ mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 Wi h 5 minu es o incuba ion a 36.6 ºC F1 + AS Sample con aining F1 a 1 x 109 PFU/mL and AS a 6.25 µg/ mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F2 + AS Sample con aining F2 a 1 x 109 PFU/mL and AS a 6.25 µg / mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F3 + AS Sample con aining F3 a 1 x 109 PFU/mL and AS a 6.25 µg / mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 3.3.6. A omic Fo ce Mic oscopy (AFM) AFM was pe o med o cha ac e ize he mo phology and s uc u e o he modi ied phages. In his expe imen , se en samples we e analyzed (Table 12). Samples we e imaged in noncon ac mode using Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 52 a NanoWiza d® 3 BioScience AFM (JPK Ins umen s). The AFM da a we e ob ained unde di e en condi ions. The samples AS, M13, F2, and F3 we e di ec ly analyzed om he p epa ed esh samples. Ne e heless, he samples M13+AS, F2+AS, and F3+AS once p epa ed we e analyzed a e 24 hou s o incuba ion a 36.6 °C. I should also be s essed ha phage F1 was no analyzed due o he ime limi a ion. Table 12. Samples analyzed in AFM. Sample Desc ip ion AS Sample con aining AS a 0.00625 mg/ mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 M13 Sample con aining M13 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F2 Sample con aining F2 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F3 Sample con aining F3 a 1 x 109 PFU/mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 M13 + AS Sample con aining M13 a 1 x 109 PFU/mL and AS a 6.25 µg/ mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F2 + AS Sample con aining F2 a 1 x 109 PFU/mL and AS a 6.25 µg / mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 F3 + AS Sample con aining F3 a 1 x 109 PFU/mL and AS a 6.25 µg / mL esuspended in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5 In sho , a d op o 15 µL om each sample was d op- cas on o a eshly mica discs (TED PELLA, INC.) in d y condi ions. The excess was emo ed. Following adso p ion o he samples (2 minu es) a oom empe a u e, he samples we e d ied using a s eam o ni ogen gas. Then, samples we e immedia ely analyzed by AFM. The images we e subsequen ly acqui ed in apping mode wi h ACTA-SS- 50 p obe (T: 4.0 µm; L: 125 µm; W: 30 µm; 0: 270.7 kHz; k: 22.187 N/m) a a scan a e o 1.0 Hz and a esolu ion o 512 x 512 pixels. All images we e ob ained a oom empe a u e. The image iles we e examined using JPK da a p ocessing so wa e and con e ed in o TIF o ma . 3.3.7. S a is ical Analysis S a is ical analysis and g aphical p esen a ions we e pe o med using G aphPad P ism 8 so wa e (Ve sion 8.0.1 o Windows, G aphPad So wa e, San Diego, CA, USA). Be o e any s a is ical analysis, each condi ion was es ed o no mali y wi h he Shapi o-Wilk es . Fo compa ison o one o wo no mally dis ibu ed samples, - es was pe o med. I non-no mally dis ibu ed, Mann-Whi ney es was used. To es di e ences be ween mo e han wo no mally dis ibu ed samples, he ANOVA es was applied. In case o a leas one o he samples is non-no mally dis ibu ed, he K uskal-Wallis es was pe o med. p Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis MATERIAL AND METHODS 53 alues o less han 0.05 indica ed s a is ical signi icance. E o ba s indica e s anda d e o o he mean (SEM. 4. RESULTS AND DISCUSSION In his hesis, he main ocus was on he de elopmen o phages ha display a p3, pep ides ha ecognize AS in cha ac e iza ion expe imen s. To his end, h ee essen ial AS-speci ic phages we e enginee ed: i) phage F1, which was used as a con ol o p o e he highe e icacy o he o he enginee ed phages (F2 and F3), ii) phage F2, and iii) phage F3. O no e, he phage M13KE (emp y phage) was also used as a nega i e con ol in all expe imen s. Thus, we used hese pep ide sequences o gene a e modi ied phages wi h he abili y o iden i y AS clus e s. 4.1. Inse Syn hesis To acqui e he h ee inse s (4554, 4554W, and 4554WN6A) ha codi y he pep ides o in e es , a 100% complemen a i y echnique was used o anneal wo p ime s, he o wa d and e e se p ime s o each inse . The inse s ob ained om his PCR eac ion should be an expec ed size a ound 55 bp (see Table 1). To e i y i he inse syn hesis was success ul, he esul ing PCR p oduc o each inse was isualized on a 3 % aga ose gel (Figu e 10). O no e, he p edic ed con o ma ional s uc u e o he pep ides was simula ed using he I-TASSER so wa e and his in o ma ion can be ound in ANNEX II. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 54 Figu e 10. Inse size con i ma ion (samples we e loaded on a 3 % aga ose gel): [A] Rep esen a ion o GRS Low Range Ladde ; [B] Resul s o he elec opho esis p ocess o each inse – L) GRS Low Range Ladde , 1) 4554 inse , 2) 4554W inse , 3) 4554WN6A inse . Taking in o conside a ion he size o he designed p ime s (Table 1), a band wi h a size o 55 bp should appea . Looking a he h ee gels in he Figu e 10 and compa ing he esul ing bands wi h he GRS Low Range Ladde , i could be seen ha all inse s showed he expec ed leng h (≈ 55 bp), as hey exhibi ed a band sligh ly abo e he 50 bp ladde band, which was expec ed. A e his con i ma ion, he DNA concen a ion o all samples was de e mined using a NanoD op One Mic o olume UV-Vis Spec opho ome e s (The mo Scien i ic), and he esul s a e shown in Table 13. Table 13. DNA quan i ica ion o he inse s and pu i y a ios. Sample [DNA] ng/µL Ra io A260/A280 Ra io A260/A230 4554 202.3 1.82 1.10 4554W 177.9 1.81 1.27 4554WN6A 213.1 1.84 1.22 No e: The A260/A280 a io is commonly used o assess he pu i y o DNA o RNA. A260/A280 a ios o pu e DNA should be a ound 1.8 (and o RNA should be a ound 2). A lowe a io sugges s he p esence o p o eins, phenol, o o he con aminan s ha abso b s ongly a o nea 280 nm. High A260/A280 pu i y a ios may indica e RNA con amina ion in he sample o DNA, indica ing a p oblem wi h he ex ac ion p o ocol. The A260/A230 is used o measu e he pu i y o nucleic acids. The p esence o o ganic con aminan s such as phenol, ca bohyd a es, chao opic sal s, and o he a oma ic compounds is indica ed by he A260/A230 a io. Samples wi h A260/A230 a ios less han 1.8 a e hough o con ain a signi ican amoun o hese con aminan s. The A260/A230 a io in a pu e sample should be a ound 1.8 - 2.2. A high A260/A230 a io could be he esul o pe o ming a blank measu emen on a di y pedes al o using an ine ec i e blank measu emen solu ion [105], [106]. 50 300 100 700 L 2 25 300 100 50 25 700 c) d) L 3 25 50 75 300 L 1 700 [A] [B] Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 55 The esul s showed ha he annealing p ocess occu ed wi h success because we e ob ained an adequa e DNA concen a ion o he samples. Rela i ely, o obse ance a ios A260/A280, i can be said ha he pu i y le el o all samples was accep able since he a ios mee a ound he e e ence alue (≈ 1.8). Howe e , he a ios A260/A230, indica ed he p esence o o ganic con aminan s likely sal s and phenols in he samples, since he a ios 1.10, 1.27, and 1.22 a e below he 1.8 ≤ A260/A230 ≤ 2.2 (s anda d alues). This could be due o he base solu ion (T is-HCl pH = 8) used in he annealing p ime s p ocess because, in i s composi ion, his componen con ains sal s, which can ha e such in e e ence in he a ios A260/A230. I should be no ed ha al hough he samples p esen ed his con amina ion, i did no ep esen an obs acle. Thus, he samples we e used in he s eps ha ollowed, he diges ion. 4.2. Phagemid cloning sys em As s a ed in he opic 1.4.1 he use o he phagemid sys em is e y applied in phage display sys em because hey o e mo e lexibili y and independence o he phage packaged du ing he ampli ica ion phase, allowing he inse ion o he en i e pep ide sequence [61]. The e o e, o p oduce he phage display pa icles o in e es , i s ly was necessa y o conjuga e he DNA o he pep ides o in e es wi h he phagemid ec o , in his case, he modi ied pETDue -1 plasmid. Display o he pep ides o in e es in p3 is achie able, bu only i he gene sequence o each inse is inse ed be ween he signal sequence, pelB, and he ups eam codon o gene 3 [58], [68]. In o de o gi e a be e design o he phagemid cloning sys em, he SnapGene so wa e (www.snapgene.com) was u ilized o execu e in silico cons uc ion o he plasmid including he inse s o in e es (Figu e 11). Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 62 Looking on he esul s ob ained in he elec opho esis gel, i was possible o obse e ha he ampli ica ion egion p esen ed a band wi h he expec ed size (≈ 323 bp) since he samples should p esen a band sligh ly abo e he 300 bp ladde band, which was e i ied. As he samples p esen ed a band wi h he expec ed size in he gel, each PCR p oduc was cleaned and pu i ied in o de o p epa e a sample o sequencing in o con i m he co ec nucleo ide sequence on each phage genome. The cleaning was pe o med using he DNA Clean & Concen a o TM - 5 ki (ZYMO) ollowing he manu ac u e 's ins uc ions. A e wa ds his cleaning p ocess, he concen a ion o he DNA o each sample was quan i ied in NanoD op One Mic o olume UV-Vis Spec opho ome e s equipmen (The mo Scien i ic), and he esul s a e p esen in Table 17. Table 17. DNA quan i ica ion o he clean PCR p oduc and pu i y a ios. Samples [DNA] ng/ µL Ra io A260/A280 Ra io A260/A230 F1 Phage DNA- clean PCR p oduc 107.7 1.87 2.18 F2 Phage DNA- clean PCR p oduc 93 1.87 2.17 F3 Phage DNA- clean PCR p oduc 64.3 1.85 1.36 The esul s exhibi ed in he able showed ha ex ac ion occu ed wi h he success. Rega ding he abso bance a io A260/A280 i can be said ha he pu i y le el o all samples was accep able since i is a ound he e e ence alue, ≈ 1.8. Wi h conce ning he A260/A230 a io, i can be s a ed ha he alues o 25 150 300 400 L 1 2 50 150 300 400 L 2 25 50 150 300 400 L 3 ① Figu e 13. Resul s o he elec opho esis gel o he PCR p oduc s (samples we e loaded on a 2.5 % aga ose gel): ① Rep esen a ion o GRS Low Range Ladde ; L- GRS Low Range Ladde ; 1- PCR p oduc con aining he ampli ica ion o gene zone 3 om phage DNA ha ha bou s he sequence o in e es o pep ide 4554; 2- PCR p oduc con aining he ampli ica ion o gene zone 3 om phage DNA ha ha bou s he sequence o in e es o pep ide 4554W; 3- PCR p oduc con aining he ampli ica ion o gene zone 3 om phage DNA ha ha bou s he sequence o in e es o pep ide 4554WN6A. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 63 he A260/A230 abso bance a io is conside ed accep able o he wo i s samples, as he a ios a e wi hin he 1.8 ≤ A260/A230 ≤ 2.2 e e ence alues, which means ha he samples a e ee om con amina ion by o ganic compounds such as sal s and phenols. Ne e heless, he same was no e i ied o he sample o he clean PCR p oduc , co esponding o he DNA o phage F3. The a io A260/A230 his sample indica ed he p esence o o ganic con aminan s likely sal s and phenols, since he a io 1.36, is below he s anda d alues. This may be due o he bu e s used in he ki because hese a e composed o sal s and phenols. This con amina ion may also ha e esul ed om incomple e e apo a ion o e hanol, which is applied in one o he s eps o using he ki . In bo h cases, his con amina ion did no ep esen any hu dle o p oceed, and he e o e all samples we e sen o sequencing. The sequencing esul s we e analyzed in he SnapGene so wa e (Table 18). Th ough a close look a he esul s, i was possible o obse e ha he sequence o he pep ide 4554WN6A was ully inse ed in o he phage genome. Rega ding he sequencing esul s o he DNA o phages F1 and F2 con aining he pep ide sequence 4554 and 4554W, espec i ely, by using he SnapGene so wa e i was possible o e i y ha he e was no ma ch wi h he sequences o in e es . A de ailed analysis using he "Align" ool be ween he esul s ob ained and he sequence o in e es allowed o de ec ha he sequences o in e es om bo h samples we e p esen . None heless, hese did no coincide wi h he o iginal sequence, as hey p esen ed inse ions. F om he ch oma og am da a o each sample (see ANNEX V) i was possible o obse e ha he ex a base pai s co esponded o unde ined peaks o o peaks o e lapping wi h he main peak (con amina ion), which ended up in e e ing wi h he clea eading o he da a. In iew o all he analysis pe o med, i was ag eed ha he sequences o in e es we e p esen and ha he ac ha he o iginal sequence o he inse s o in e es was no de ec ed was due o e o s associa ed wi h he sequencing p ocess. Thus, i was con i med he p esence o he co ec 4554, 4554W, and 4554WN6A pep ide sequences in he espec i e DNA phage sample. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 64 Table 18. Sequencing esul s o DNA phages. In unde line he egion he sequence o he pep ide o in e es , and in in ed e ased he addi ional inse ions in nucleo ide sequence o he pep ides o in e es . Phage Inse o In e es Re e se Pep ide Sequence (5´- 3’) Nucleo ide Sequence om sequencing (5’-3’) F1 4554 GGTGGCCACGCCGTGCAC CACGCCCTCCTT …TAATTCTTTCTCACAGTAAGCTCCGTCGTACGG GTGGG157CCACGCCGTGCACCACGCT176CCTCCTT CTAATCTAC F2 4554W GGCCTTCACGCCGTTCACG ATGCCGTCCTT …TCGACGGGCCTTT161CACGCCGTTCACGA A176 T177 TGCCGTCCTTAAATCA F3 4554WN6A GGCCTTCACGCCGGCCAC GATGCCGTCCTT …GAAGCTTAGTCGACGGGCCTTCACGCCGGCCA CGATGCCGTCCTTAAACGACTGTCGGCGTGCTTG … 4.4. Cha ac e iza ion assays The cha ac e iza ion assays pe o med aimed o de ec whe he enginee ed phages displaying he pep ides o in e es (4554W and 4554WN6A), conside ed in he li e a u e o be able o in e ac wi h AS in he 45-54 egion, we e indeed able o ecognize AS [34], [94]–[97]. Fu he mo e, hese assays we e in ended o mo phologically cha ac e ize he syn he ic phages p oduced. 4.4.1. Physicochemical cha ac e iza ion o he phages pa icles and phages pa icles incuba ed wi h AS Fo a phage concen a ion o 1 x 109 PFU/mL and an AS concen a ion o 6.25 µg/mL, phage pa icles (M13, F1, F2, and F3) and phage pa icles incuba ed wi h AS (M13+AS, F1+AS, F2+AS, and F3+AS) we e cha ac e ized a he le el o hei size dis ibu ion (Z-a e age) and Ze a po en ial unde di e en condi ions. The esul s om his i s es , using he Ze asize Nano ZS equipmen , a e illus a ed in Figu e 14 and Figu e 15. Acco ding o he esul s p esen in he Figu e 14, i was possible o obse e ha phages wi hou incuba ion, wi h he excep ion o phage F1(≈ 446,43 nm), p esen ed he expec ed size since he size dis ibu ion ob ained o phage was in line wi h he li e a u e o phage M13 (≈ 800 - 1000 nm) [58], [68]. Besides, phages F2 (≈ 805.07 nm) and F3 (≈ 811.65 nm), which display pep ides 4554W and 4554WN6A, espec i ely, showed a la ge size compa ed o phage M13 (790 nm), which also was expec ed. Only phage F1 p esen ed a smalle size han expec ed. As al eady poin ed ou phages F1, F2 and F3 displayed pep ides a he p3 p o ein end. Howe e , i should be ema kably no ed, ha he Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 65 pep ides o phages F2 and F3 (4554W: KDGIVN6GVKA and 4554WN6A: KDGIVA6GVKA, espec i ely) a e qui e simila o each o he di e ing only in he amino acid a posi ion 6, whe eas he pep ide o phage F1 p esen s an amino acid sequence wi h mo e dissimila i ies (KEGVVHGVAT). This pa icula i y highligh ed he e can jus i y he size p esen ed by phage F1. In o he wo ds, pep ide 4554 may be in e e ing wi h he o e all phage a chi ec u e, hence he phage is smalle in size. Conside ing he da a o samples incuba ed o 5 min a 36.6°C (condi ion 1) i was possible o obse e ha he e we e no signi ican changes be ween he non-incuba ed and incuba ed phage condi ions. Fu he mo e, when compa ing samples con aining only phage wi h samples con aining bo h phage and AS, i was no able ha he e was no subs an ial change in he size o phages when incuba ed wi h AS. A e 24 hou s o incuba ion (condi ion 2), a dec ease in he size dis ibu ion o he samples con aining only phage was no iceable, whe eas he samples con aining bo h phage and AS, wi h he excep ion o sample M13+AS, showed no size luc ua ions ela i e o he 1 condi ion. Finally, i was e i ied ha he samples, excluding he sample F3, p ac ically s abilized hei size a e 48 hou s (condi ion 3). A close look a he se o in e ences d awn om hese da a aises he hypo hesis ha phages exhibi ing a pep ide in hei p3, ha e es ablished some kind o in e ac ion wi h AS and ha his in e ac ion does no in luence he physical cha ac e is ics o he phage, as h oughou he incuba ion ime he phage has conse ed i s size. Ne e heless, o his hypo hesis o be co obo a ed o he ypes o assays we e ca ied ou . I should be unde sco ed ha hese alues a e only an es ima e and may no be an app oxima ion o eali y, since o he measu emen o he sample sizes he phages we e conside ed p o ein ma e ial as a whole. In addi ion, he equipmen gi es a sphe ical app oxima ion o he size. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 66 Figu e 14. Cha ac e iza ion o phage pa icles and phage pa icles incuba ed wi h AS p epa ed in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5. The esul s plo ed show means  SD o n= 8 samples. Size dis ibu ion o e ime ( 0: samples wi hou incuba ion; 1: samples wi h 0.083 h (5 min) o incuba ion a 36.6 °C; 2: samples wi h 24h o incuba ion a 36.6 °C; 2: samples wi h 48h o incuba ion a 36.6 °C). S a is ical signi icance o he da a was de e mined by - es and wo-way ANOVA (*p ≤ 0.05 and ****p ≤ 0.0001). S a is ical analysis be ween he samples o each ime poin was e mina ed by wo-way ANOVA (da a no shown). Rega ding he Z-po en ial esul s (Figu e 15), hei analysis showed ha nea ly all samples exhibi ed a uni o m loading pa e n o e ime. In he 0 condi ion (no incuba ion a 36.6ºC), whe e only he cha ge o he samples wi h isola ed phages was e alua ed, i was isible ha he phages ha displayed a pep ide a he end o he p3 p o ein (F1, F2, F3) showed a mo e nega i e cha ge han he phage M13 (con ol), which indica es ha he pep ide a ached o he p3 p o ein con e ed a mo e nega i e cha ge o i s espec i e phage. The pep ides exhibi ed may be al e ing he phage s uc u e, p omo ing a g ea e exposu e o he nega i e amino acids on he phage su ace, which induces his inc ease in cha ge. In he emaining condi ions and compa ing he esul s ob ained, i is concluded ha he incuba ion o he phages wi h p o ein does no in e e e wi h he s abili y o he phage cha ge, and ha ega dless o he incuba ion ime, he Z-po en ial emained p ac ically unchanged. The sample wi h indi idual phage M13 p esen ed he g ea es oscilla ions, wi h a signi ican inc ease in cha ge (in absolu e alue) du ing he cou se o ime. I should be no ed ha i was expec ed ha he phages would show a nega i e cha ge gi en ha he ou e laye o he phage p o ein shell is mainly occupied by he nega i e N- e minal egion o he p8 p o ein, which is highly ich in acidic amino acid esidues ha in e ac wi h he sol en and endow he F3 (4554WN6A) 0 200 400 600 800 1000 0 1 2 3 Z-a a age (nm) Time (h) M13 M13+AS F3 F3+AS F2 (4554W) 0 200 400 600 800 1000 0 1 2 3 Z-a a age (nm) Time (h) M13 M13+AS F2 F2+AS 0 1 2 3 0 50 100 150 200 250 500 1000 1500 Z-a a age (nm) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) ***** **** **** **** **** **** Samples: 0 1 2 3 -30 -20 -10 0 Ze a (mV) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) **** **** **** ** * *** **** ** * Samples: [A] [B] F1 (4554) 0 200 400 600 800 1000 0 1 2 3 Z-a a age (nm) Time (h) M13 M13+AS F1 F1+AS Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 67 phage wi h a low isoelec ic poin (IEP) [107], [108]. Paolo e al . p o ed ha he cha ge o phage M13 came p edominan ly om he cha ges o esidues cons i u ing he majo i y coa p o ein, p8, and expe imen ally de e mined ha phage M13 had an IEP o 4.05 ± 0.065 [107]. Thus, since all samples we e p epa ed in a bu e wi h pH= 7.5 and gi en ha he cha ge o p o eins is ela ed o pH and IEP, i was expec ed ha he phage-con aining samples would be nega i ely cha ged because pH > IEP [109]. Figu e 15. Cha ac e iza ion o phage pa icles and phage pa icles incuba ed wi h AS p epa ed in 20 mM T is-HCl + 100 mM NaCl, pH= 7.5. The esul s plo ed show means  SD o n= 8 samples. Ze a-po en ial o e ime ( 0: samples wi hou incuba ion; 1: samples wi h 0.083 h (5 min) o incuba ion a 36.6 °C; 2: samples wi h 24h o incuba ion a 36.6 °C; 2: samples wi h 48h o incuba ion a 36.6 °C). S a is ical signi icance o he da a was de e mined by - es and wo-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p≤ 0.001, and ****p ≤ 0.0001). S a is ical analysis be ween he samples o each ime poin was e mina ed by wo-way ANOVA (da a no shown). F3 (4554WN6A) -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0 1 2 3 Ze a (mV) Time (h) M13 M13+AS F3 F3+AS F2 (4554W) -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0 1 2 3 Ze a (mV) Time (h) M13 M13+AS F2 F2+AS F1 (4554) -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 0 1 2 3 Ze a (mV) Time (h) M13 M13+AS F1 F1+AS 0 1 2 3 -30 -20 -10 0 Ze a (mV) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) **** **** **** ** * *** **** ** * Samples: Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 68 4.4.2. P o ein elec opho esis in e p e a ion As p e iously men ioned, he esul s om he analysis wi h he Ze asize Nano ZS equipmen a e no su icien o alida e an in e ac ion be ween he designed phages and he a ge o in e es . To con i m and e alua e he phage ecogni ion capaci y, he samples used in he p e ious assay we e submi ed o an SDS-PAGE analysis. This p ocedu e aimed, by means o he in ensi y o he bands ob ained, o e i y which o he phages showed g ea e binding o AS. Thus, based on he s a ing poin ha all samples con aining phage and AS had he same concen a ion o p o ein, eco e ing he ee p o ein p esen in hese samples and loading i in o he gel would esul in esponses. The mo e in ense he AS band (6.1 kDa) o a gi en sample he lowe he binding capaci y be ween he phage and he AS. Un o una ely, as seen in Figu e 16, he SDS-PAGE p ocess did no occu success ully. No clea , well-de ined bands we e obse ed on he gel, ende ing in e p e a ion o he esul s impossible. Al hough wo s aining me hods we e used, and despi e he ac ha he sil e ni a e s ain was mo e sensi i e and highligh ed new bands ha we e no isualized in he gel when s ained wi h BlueSa e, he bands we e mo e smea ed. This made eading he esul s e en mo e di icul . Samples 1, 3, 6 and 8 we e used as con ol o samples 2, 4, 7 and 9, espec i ely. E en hough he lanes o hese samples showed bands, hey we e no well de ined. In addi ion, in samples 2, 4, 7 and 9 simila band pa e ns o he espec i e con ol samples we e isualized, when only bands be ween he 5 kDa and 10 kDa bands co esponding o he molecula weigh o he AS monome s (6.1 kDa) we e supposed o be obse ed. The p esence o all hese bands means ha he elu ion o only ee p o ein did no occu success ully. I is no ewo hy ha he ladde band pa e n was no well de ined and sample 5 (con aining only AS), used as a posi i e con ol showed a he ain and unde ined bands. In addi ion, his also did no show he 6.1 kDa band, which was expec ed. This e idence would in alida e any kind o illa ion. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 69 Figu e 16. SDS-PAGE gel: ① Rep esen a ion o PageRule TM Uns ained B oad Range P o ein Ladde ; [A] Gel s ained wi h BlueSa e and [B] Gel s ained wi h sil e ni a e (L- PageRule TM Uns ained B oad Range P o ein Ladde ; 1- sample wi h eco e ed p o ein om M13 phage sample; 2- sample wi h elu ed p o ein om sample wi h M13 and AS; 3- sample wi h eco e ed p o ein om F1 phage sample; 4- sample wi h elu ed p o ein om sample wi h F1 and AS; 5- sample wi h AS P o ein om s ock; 6- sample wi h eco e ed p o ein om F2 phage sample; 7- sample wi h elu ed p o ein om sample wi h F2 and AS; 8- sample wi h eco e ed p o ein om F3 phage sample; 9- sample wi h elu ed p o ein om sample wi h F3 and AS). Finally, i emains o be men ioned ha om hese esul s no conclusion abou he binding capaci y be ween phages and AS was ob ained. Consequen ly, no conclusion abou he pe o mance o each pep ide was d awn. The absence o esul s can be explained by he insu icien mig a ion ime o he p o eins in he gel, which caused no de ined bands o be ob ained in he gel, and by he lack o dena u a ion o he sample p o eins. Be o e being loaded in o he gel, all samples should ha e unde gone a dena u a ion s ep a 95°C o 5 minu es so ha he samples would show a band on he gel co esponding o he na i e o m o he p o ein and no also show a ange o bands co esponding o oligome species, which hen end up in e e ing wi h da a eading. In addi ion o hese wo ac o s, he columns used o he p o ein elu ion may no be in good p ese a ion condi ions, which may ha e condi ioned hei pe o mance in he elu ion s ep. In summa y, he SDS-PAGE p ocedu e should be epea ed again conside ing all he condi ions lis ed he e. 15 ① 1 2 3 4 L 5 6 7 8 9 250 20 10 L 15 1 2 3 4 L 5 6 7 8 9 250 20 10 L [A] [B] 5 Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 70 4.4.3. Cha ac e iza ion o AS-speci ic phages by STEM In o de ed o ob ain in o ma ion abou he mo phology o he indi idual phages and phages incuba ed wi h AS, he 8 samples shown in Table 11 we e isualized by STEM, as shown in Figu e 17. F om he SEM images ob ained, i was no possible o de e mine he phage mo phology and how i is a ec ed upon incuba ion wi h AS. Consequen ly, no conclusions abou he binding capaci y o he enginee ed phages could be d awn om his assay. The low esolu ion p o ided by he equipmen and he exis ence o sal s om he solu ion used in he p epa a ion o he samples we e p eponde an ac o s ha made i impossible o cap u e de ailed and clea images. Thus, i was ag eed ha a new echnique wi h highe esolu ion, such as ansmission elec on mic oscopy (TEM), should be used o analyze he samples a he nanome e scale. Fu he mo e, o a oid ha he con en o in e es o he sample is masked by sal s, a new bu e , con aining less o no sal s in i s composi ion, should also be used. 5 minu es o incuba ion a 36.6 °C F1+AS 1 µm F2+AS 1 µm M13+AS- Con ol 1 µm F3+AS 1 µm Wi hou incuba ion F2 (4554W) 1 µm F1 (4554) 4 µm F3 (4554WN6A) 1 µm M13- Con ol 1 µm Figu e 17. STEM esul s. STEM images o samples wi h phages (M13, F1, F2, and F3) and samples wi h phages incuba ed wi h AS (M13+AS, F1+AS, F2+AS, and F3+AS), wi h and wi hou a 5 minu es incuba ion a 36,6ºC. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis RESULTS AND DISCUSSION 71 4.4.4. Cha ac e iza ion o AS-speci ic phages by AFM Visual cha ac e iza ion o he AS-speci ic phages was also pe o med by AFM. In his assay only he mo phology and size o phages F2 and F3, which exhibi he pep ides wi h high po en ial o ecognize AS, 4554W and 4554WN6A, espec i ely, was compa ed wi h he wild- ype phage M13. The AFM images a e shown in Figu e 18. 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B own e al. , “An easy- o-p epa e mini-sca old o DNA o igami,” Nanoscale , ol. 7, no. 40, pp. 16621–16624, 2015, doi: 10.1039/c5n 04921k. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis ANNEXES 85 8. ANNEXES 8.1. ANNEX I- P ime s Sequences The p ime s sequences used in PCR p ocess and in he samples sen o sequencing. Table 19. De ailed in o ma ion abou he p ime s used. P ime Sequence 5’-3’ In o ma ion Ta ge / Templa e #30 (Fowa d) ATCGATCTCGATCCCGCGAA P ime s used o sequence o o con i m he inse ion o he sequences o in e es in o he plasmid Annealing wi h p ime 6 o p o e inse ion o AS inse s in o he pe Due #6 (Re e se) CTAGTTATTGCTCAGCGGT P ime s used o con i m he inse ion o he sequences o in e es in o he plasmid Annealing wi h p ime 30 o p o e inse ion o AS inse s in o he pe Due #25 (Re e se) CCGCCACCCTCAGAGCCACCACCCTCATTTTCAGG P ime s used in he PCR p ocess o ampli y he egion o in e es o phage DNA ha includes he sequence o he inse o in e es To con i m co ec cloning a e in ec ion wi h M13 Helpe Phage. Pai wi h 23, 24 and 41. #41 (Fowa d) AAGGACGGCATCGTGGCCGGCGTGAAGGCC P ime s used in he PCR p ocess o ampli y he egion o in e es o phage DNA ha includes he sequence o he 4554WN6A inse To con i m co ec cloning a e in ec ion wi h M13 Helpe Phage. Pai wi h 25. #42 (Fowa d) AAGGACGGCATCGTGAACGGCGTGAAGGCC P ime s used in he PCR p ocess o ampli y he egion o in e es o phage DNA ha includes he sequence o he 4554W inse To con i m co ec cloning a e in ec ion wi h M13 Helpe Phage. Pai wi h 25. #43 (Fowa d) AAGGAGGGCGTGGTGCACGGCGTGGCCACC P ime s used in he PCR p ocess o ampli y he egion o in e es o phage DNA ha includes he sequence o he 4554 inse To con i m co ec cloning a e in ec ion wi h M13 Helpe Phage. Pai wi h 25. #7 (Re e se) CCCTCATAGTTAGCGTAACG P ime s used in he samples sen o sequencing To sequence o ampli y he M13KE gene 3 a e pep ide inse ion Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis ANNEXES 86 8.2. ANNEX II- P edic ed con o ma ional s uc u e o he pep ides The con o ma ional s uc u e da a p o ided by he I-TASSER so wa e: Figu e 19. A schema ic ep esen a ion o he I-TASSER p o ocol o p s uc u e [99]–[101]. Enginee ing O Speci ic Bac e iophages Fo α-Synucleinopa hies Diagnosis ANNEXES 87 8.3. ANNEX III- Expec ed Size o PCR p oduc s by SnapGene so wa e The ac ion, PCR, o he SnapGene so wa e was used o pe o med in silico he expec ed size o PCR p oduc s: Figu e 21. Expec ed size o PCR p oduc o ampli ica ion o gene 3 zone con aining he inse sequences: #30 – owa d p ime and #6 – e e se p ime . p oduc _PCR_#Pe DUe _g3_pelB#p ime s6-30 1840 bp Figu e 20. Expec ed size o PCR p oduc o ampli ica ion o gene 3 zone wi h p ime s #30 and #6 (nega i e con ol - emp y phagemid): #30 – owa d p ime and #6 – e e se p ime . Pep ide o in e es gene p oduc _PCR_#Pe DUe _g3_pelB_inse #p ime s6-30 1858 bp