Circular RNA analogs comprising 7-methylguanosine cap, preparation, and application thereof

Abstract

The presented invention relates to a novel class of nucleic acids, which have circular topology and contain at least one unnatural internucleotide linkage containing a 7-methylguanosine cap analog.

Description

[0001] ummar oenvenon conearnes aeneeas onnveen unonnau reraaenseorn auc neoovee canssa oe n cuocec acs, wcave crcuarooo an man oecs oescosen nann a-meuanosne ca anao.e sness o′-moerecursvoernsonre a-rcer acnaoss o an crcuearr suseucenrc crcuaa are oane e reacon.e crcuarzaon occursu rzaon va a cemca one eoeresence owo cemca reacveunconarous,rans acrona′ e,n w aenrea asnoee rrea acveer′o eun. aeeu′n ecnonarou ae′ ensncororae co-nvenon encomasse sncororaeos-ranscrona.e s encon ars s oerenensrom no n,ncun messener sransaon w an eoceennc.ee crrcan csem enccaon un amoroeen c acrccorns ooes sanvenon unero oe emomens oenvenonscoses crc me seuence.ne conann aunconaze anaosa are oaneromre-crcs cas are more ecenran-smaeeaunan corscne cas o anaoe sa ame seerue′n ecne. wuc crcs conann rearaon o crc anaos, meos oreara oue ca.eos o rearaon o aroen on o corresonnre-crcs, meos o eneeraes an vacc onraoens aere, a anso asccoaseo.n oescose crc anaosne conex o e oenvenon meeoresse oneronuvcenno anr roeeanes oroee mno vecrou,esn, meos oroucn crcuar moecues, orrevenn asease. ces, orvn oransms, an meos orean acroun oenvenon eenseeevsern.1erenerao, s one oe mosoen susancesne conex oeraeuc meos o enc oeraeuc messener m semsromo avance m sremavrer aanre ceemca ex moonucecaaseosne o..ee mcan csoem.eoxesn serum anne coso2, rcuarzaons one oe mosr ,eaenases, exosomes. eson.3oecve mocaons o m, asrevens exonuceoc reuarcreonns,6-9n aonwn re maceoonss ca oaze cr ccauaarzca soenu aernease on cemca reacons4, 5, e1n0z-13mac crc . ces emeen crc recursors re- e re seorueenc mese, coems o crcuarzaona arease on cemca reacons re one use o sor or′-amno , wcca un snesse vaosorame meo, suc as′osorae meamnorocaero conensa4,o5nnuce canoenromer or-e--- acss a rous meor ooromuce . eosorame so ase sness o nucec raccaoreroucon o on o moe an unmoe oonuceoes,owevers no ese meos enco we rce on n seuences.n conseuence, crc oane w roen oree, a arvee usu saor s senuecnacnes anonaesr.me usene conex o s encon a oarseeove.6r,,7, 14o crc eunzarmzaeso rne cuanree′ c marorneo ouos w ases, eseca ase an suose enuzenmes araee ca wae oen′ramo oece rose aorea′e enr oecu arsorse,r wenc or aree sarmse aena seran an.15e′ en oe same sranowuaraoncrcuarzaon o reacon occurs w en an s11, 12, 14, 16ever,eaveeenounoe sroneenen onare o suc enzmes.euence. ssermena, as cuorec ormroveerormance e ae m.oasve re ccorcunasreze meos on vro crc uusanrzaeonernmvuoeve cnaraonc nucec acs.8-12, 17esseoe se-scnnronc seuences.11, 12e ssem co -exon ssem an omzenron seuences.10, 17urne se- nsss ouseara exonsane a- roxrou scn reacon,woransesercaon reacons occur.rs,′ en oenerm oe aauea ennosanees nuceeo′ se ecne saee,s w′ scc rees sues.ne ccorncu,arezareoen.roxrou ae ce e a.av rrcouzamrzeasoan.e8so soow nrann naeooe u-esxwr ues nsesre-o eror aam unsz oce craas cconu cara eane va an overexre e.sea osso vesnn ′e-reosrna roaon sesemorwser osaeermn, wc areenae e asec. x an′,′-ccc evee soo-maer-nre,oreereo mree caonnsoasee us oen c oanare-cc nrcucec a oc an acvven r seeuureenc see.ueunrceermesoren, an crc’s a o unero crcuarzaon canenere eresence o cemc re- suc as naura an snec nuceoses an nuceoases.12a mocaons, exnere ossone.ees maanws oe cnrecs o eunareo cocnerxan osaeonneae mavcerne arn areneeraessowroen mecansms.18, 19one oescovere or ease on ca-eenen as-meuano snesse crcuar20, 21crc conan eemens suc srucure,eranssaneon ca o, c ourrr aennnnow on c′r ncuceos occeu arsnano an. ueneo crcuar an “ca-ess” eenen manner.5, 7, 9, 11, 12, 19, 22, 23n euaroc ces, ca-eenenransaornnas r muocsom meor een ere scev- anseuenc-e,e cerncensr eaxnsao sn.19ncnane e con o oneerraan-,vese an saero oen c ercxressons er,n msassoner.12, 14romese meos oreamens.cosaerms cacrecuuar s anranser veces, aon w reae comosons an s an maeras useunrocauc cnomoesroencsur csoomr orrs cnrcu suacr crcusaraveese ans a wse asrecursor ceocumen. oscosen asransaaescer ores ao voeccoaro arc mvaenns cerc euuaarroc, c aeos.wneroucon o a crcuar recomnan esxcoressesso an re vceocmor,nanre-c nurccueacrz aec moecue oeranscrona crcuar , armaceuca cocmueoss ao cnr acnuarroe , crcuar , recomnanos ce, nucec ac moe n wrecarn con maenso a. secercanscr eoenmeron.uc oe recomnan ase.scoses ccc an a meoorrouconereo wc comrsese use o srounceu ore wenrevrooeusn-co renor cerc meo.s are suaeorncororan a-meuanosne ca ummar a ceemrescean reasccoosnurceermovcaes c nrocvuear anzaaoons o o crc, resarae csasn oeere carrcerom seruee-nccrec,es va sze.ueo cemca naure oe crcuarzaonrocess, ena n or meuanosne ca wne srucure o crc.nexe esncororaon o- wn a crc anao moecuencre ce,eresence oe ca srucure unmoe crcs oe asessransaona acv,n comarsono cemcao crcsa uneroran ssamaeo sne vuaen ccae.-eeerneoerne, mreecseannsmsc.osureves unreceene access e a suresseenuensc coseumrecaro cvrcuesar az carc anaoreare n vroranscron reaconoowe oroen enzmes or caac nuceocn ac reac seonu,e wnceesr.en sa crcuarzaons oanene asence sruecurerese onrosc aosu-mreerovueasno as cnrec ca an aanoao srucau croen anan asn eneerrn aa r-moesomau eannorsn see c sae auneancoe. eresenscosureroves a crc anao accornoormua: w n aser n aenn: eacne oer wnsn a vaueromo an eacneenen ,3,2, or an eac o onssnneenen or an eac onsneeenen, or an eac onsneenenen, or ,′ an eac onsneeennen, a na, aura,,-a oraoen -mse aneuaarn oorsnrean ccae srucner, weren sa mnoere c oorm unrnsaesur oa n au ncaeuorsa,e moasee or unnaura n some emomens,enureer.s a srucure accornoormua,ormua orormua, we easre-nm:euanne, naura or unnaura-meuanneervave or anao, qc1, oq2,1q3rosune3ensneneen,en3, o,r or, weren1+2+3s eua or qq13Lasnq2sneenen,,2,aoen or aoen Ls a, sn,e c,2,aoen2or aoen2Lss a sne ceemmccaaoonn,,,, or, a,nea2r o orr arannear orrancener me, em,o-meen,2cener n so s,-ae,ne-rsuss a sureucuare ac.cornoormua were eacs o-mn:erouuanne,s nanureae onr uennnaur,a o-rm,e weruanneervave or anao, ,,1sne3enen,, o en1+2+3s eua or qq1q13asnq2 q3,q2,sn,ee2n,enaoe,n2o,3r r2,aoaeonen2or aoen anLL1nL3asn a sLn2aree cnemeceanenon,,, ,,-,e,2o-ra an,ear- orsursancueeaner, weren aeas one oL1so amnL2s e emLn4o aoremneense,neenn serne co cmemrsceas oo anse,e2or anear orrance ner. n some emomens,ener s a srucure seececa ameonae o.oownormuas: n caeracreerseznescoasu are crcrove asna ao merecourso or sornmuesas o crc anaosescre aove, w n =eren: an- eac o11rounnsneenen ,3,2, or a ann e eaacc o o1rroouunssnneeeenneenn, or or , a ann e eaacc o o11r s a′ annea erac o o1oruou rounnsnsnn see nenen eeennen en,, a na, o ar ura,, mo-ae o orr uanonaeunra nuceosease aveeenrersenraenc aeovenerreera eners a srucure seece amon oormuas wcsrs oxzeurn se oe meo,en aaeeervaveormua, w n =ere an-n: eac o11rounnsneenen ,3,2, or a ann e eaacc o o1rrounsneenen or an eac o1oruousnnseneeneen, or , an eac o rou sneennenen,,, o ar,- s a′ annea erac or o1ran1cerounnernsneenen a naura, moae o orr uanonaeunra nuceosease w crccs n aenxaon.cuaen reucve envronmenurn se oe meo,eneesre on soem mee emoosm ceanrrse, s oeun ane sreesence a ore caarre, oureenr oane re aac ao cr.n some emomens, seemeraureeow°.n some emomens, s4e oe meooncensr caaroneow m, a a souon,reera a –,nresence oa eow m.n somree em ouonm aenus,e sree o oorm seera os coem meemoen araer caror ae ounrerceusersnoc3e o a naura, moe, or unnaura nucec ac a r seuence.n some emomens,e crcnna aocososoaerome reacon mxure anown meo o soaon, suc asrecaon cromaor,a sze excu.son cromaora,e eecrooress, or -erormanceu n verroes seenu.scosureroves a use o a crc anao escre aovenroenrouconn an meeore,sennarsccousaurreorrovroeesn a crorcucon anna aonne vscvro seeu a.oveor usen aeraeuc oranosc esecrreese anoves.cosureroves a armaceuca comoson comrsn o a crc anao enons onnee oss senen oe aerrwos we, acermss unsveenoerneneoanves.e same meann as are common unersoo oreeeorxmsr “nouceercva acve”s a ann “n ouc nec ac omer” reero aomerc susance comose o ro nonc aura occurrn or snec nuceose monomers,ncun nauraano onrca snneaces an an aneree o, aos wmeerz aaso nnu.ceecse acrsas ceosmncoue,u are nomeo, oranoosae, seenoosae, an osonae m se o osorooae,oce nucec ac ,e e nucec ac , anoereeso,s me nourceocn aoc nucec. acs , ane ceremmsc “amo moecaona”, m “amoe anacrou”, an “mo e”ne conex o nucec acs reero oo, aoacosmeane o o a neu mcoec acn moroeue cueoo .e a s nuucaer,c n aucceo maoseec,une.ernucsenocuees,osuas ne,o orm′-e′- moenoemrmers “ one arn nuucceeoc acenoaem”e rre.erso a covaen or non-covaenon oronsa connecwo moeeerms “nuceosease”, “nuceoase” an “nroenousase” reero aar o nucec ac ancnureasm.oeecu rearerrneer nauccoenos.asese m raeerreenv novenasearn,ase sacn, an oerner- mo e, unnaura or uceoasesncue,u are no me o naura, -me snec urnesuc as aenne, uanne, oxanne, xanne, mne,ua unraocsn,e, -mreouracae,nos-nmee, c-meosnea,eno-snerox anmermconsene,suc as cosne, me seuourne, nconame nuceoases, as we seuourne, -susue aenne, -su as-amnourne,,-amnourne,avn, -eazauanne, -susue ursnuee, -suuansneu,e c-suossne,ue- an-reaczacacen nunce,eoas-essu.sue seeuenecrme”s an “nu “cseeuc ac seuence”, “nuceoe seuence”, “nuceose seuence”, “nuceoase ac moecue. ence” reeroe orer an comoson o a seres o nuceoases w n a nucec conesesrmns “ onucweoo nuasceeoaar”se asno “unaseoa ear”c re oerero aunarmoeennao unns o. oeuere-srane nucec acso secc roeen.. wononeeasweeeanr nnuc,eoooasse m “asearn” reers or non-canonca es,naase canarne c.anonca ason-rc asearn caeraecrmersz “e rzaao,n”a an u “nerr azer”o reraereo x c,on anerac roenosn o exsw , ano nc or -m nu m ocoere ,c nu a accec ac srans. s a seuence-eenen srucuree.. aouee -ua srruanesx. manan ore nuecrmec “c aocms.emenea crom” reeemrseeno -a r erea e sneon eunene scw reusee c on ur s re seuences owo or more srans o a nucec ac rzaon anorm a seuenc .ranes co amre cemaerancaerrse caneua orear unae.ro moeeceurme.s “nneeaarr” nou acn meec “c rs acrcu wsa ar nr” aen uran ceoan orex r mo o nucec acs reeroeooo o a nucec ac nucec acs are omeers wna neoenweaene′e anween′ en′ asn. ′ ens. rcuar oemeremrso “ ac crcrucuarazra nuocne” acn ac “ccozmaeor.n” reero arocess oransormaon o anear nucec ac uneererom csr “ccuracrzaonreecaurnsor”o a an cr “curea-rc nruccec a”c reeorom aer.near nucec ac omera can ceemecrma m “oneere”sna co cnoevxae on crcounar′ n auncec′ e ancss o reeersoo amner n.aureae orrm s “nneerc” cnocmunesaon o nomeo an near orrance comnaon o a , aen, a n, e ,us caramae,razo, osoeser, a er, eser, ame, amne, seece amon ooown srucurmes:oosae, or osonae moees.narcuar cane were n ms a naura numerrom o . nenerm a n “acaura an oaro m”o reeers nouc aneos coems,o nuunceoae cso,n ora nnusc neacu arac osr. moeeer′-′-ooosaeo canonca, as we as non-canonca ca srucure m “a anao” reers -ca, -ca, an -ca, as we as ca an srunccuures,us nomeoa,a,a, rose, or nuceoases.narcuar, cane see es moe wn ooosaere, cen suecescron, esseca ce amon o srucuresescren anocumen , ,: , , , . , , an coneaenrms “-nmaeura, muaonosene o orr unsn naauurara- omre moueanosne ca srucure” reerso an comounao ca anaos suc as m7, m7, m7,aerv,aave., aneeramn.cues,us nome eer aerms “nacernaae r caos-omnae eennren sen” aano “n ora”n rseaeronone sreacueonnce osran anre caracersenvovenormaon o rosomes anransaon.eerm “ seue ceuarroens meonceaomocars vrus ,oxsacevrus nce”ncues,, resurs no snca vrus m6meaon ses, vra seuences,uman seuences,a mo6r m seeuenaceso.n aeesr cmos1m “1,ses, seuences conann m6mocaon, e aamers, an arca r1ea2mn, 2s2ec, 2nr4arono” a or “ranscermaone. rearcanoscnr” reero meosor enzmac sness o arca seusasnn vroransc on can occurn ces,n ceuar ssems orn eenen omeras ron reacon.ranscrons carre ounresence o a - reacon,e seuence oe,e ronuceeoserosaes s, an a emae.urne ona,eranscron caneerm cmarnra aee ors os coe an amenorm o moecues.ranscronnaors,ranscron ,u or mnoeer neusecnecoes oe arosonaae rse.aens, suc as comeoerumnss “ anaeor” aanrc “ese,ecaa meae” reero an comoun,arce, or an comnaon o aare moecue covaen aaerec ornreceece.eae cane assocae wae, snae, a cr nor non-covaenneracons.eae cane a raosooe,o2,13,15,32,19om,o canornee,s a,uuoorroesceornee,s a, raoeanm.nees, creouemrraernaes,eenrcueneessus nome nroes, azoes, ,on,esoon,oc ac an-aceaacosam, nnae.ames, ceeavaermes o “ caerroona–cear oxonaoonn”s, o “c vs-cnoa o cxs-aoosn”re asnen “rnosee s orxuaon” reero arocess o nucec acseano aaeeervave o a nucec ac. cure o a naura or snec crceuaerrmzas “orne reucerveo a ammnnaaoonn” rea acnon “amnvnoavonn” anaee coneexerv oa nvuec oe ac n aucce mc aoc.caon or eee rremer “rreeuc revuec envvronmen” reerso conons uner wc cemca reucon reacons occur. canoorore, soeum envorroonmrene,ncruneseouranes, o nro soumm aescoroae.eresence o soum eerms “ao” an “aoen” reero aaoens, suc as coro,uoro,romo, anoorous.nceueerms “uas n”o reemrseo ao sn meeo,n e ca,nr oo,rsoocarroons, r na-nun,r soemc-uo,so caruon aoms an en,soam,ex, oc, anoecanrous. ,er-u, -meeermox “,-a-eo”x,eno-ersooex,rou-so -ro,o wxe,re-uosx an, a-so,u anoxn,c anues-eunosx noroums.eo oe-erms- “e-ene-amn m,e o w mo rner ae”, “ orms mo”, “ rou”, an “” reero a cemca mo reconse2n anon2c2or2o an a s sau.suen.rucure oe mo ma as wee ore′ eenrm “raea ccanv reeaucnc wona anoroeur”sa remeer osro aneunconarouasncororae ae ′ o oernaura or unnaura cemca moees creonna rneeac wvernou srunc aur see oecvee c mracnnernrerceusersnoce e reresenavears o′ an′ reacverous wc are reacveowa r. are nomeo anaee,raze anaee, azres a enac a oenr,ncueu ccoocneervave, amno an caroxcrou, amno anuo e, aze an osne, ec. rosuonrou, aze an reeceramosn” “n reucereco acesroc aecoorecaon”, “nucec acsrecaon”, an “acoo nucec ac ss o nucec acsoaon.s aecnueoesa an concenrae aceone ans sraoms ae.ueo.u sso soumuo ancse.ae, amomno onumoa arce oraaen,c souvmen cse.. eano,soroano, umercoraeo nucec ac souon causeserecaon o nuceorc aec, sso.umercorae, oeoermcas “ maocroamcroaecmuees aecc eorecrooress” an “ ” reero aecnue useor searaon o uncon oe mass, conor noer eecroorec mo.ecroorec mo s a enaurn conon maon,ooo, an care oe moecue.en serormen same. s,rovesnormaon one comoson oe nucec ac secesne aeecnermuse “oreverse-ase ressure cromaora”, “- , an “ ” reero mxure. rr searaon,encaon,uancaon, ansoaon o comonens o a same an souonees onums consun aow oressurzeu moeasea mxure o sovens eren ws mrooue a an so sa marx oe saonarase.omonens oe samenerac saes.ssacae ao cnoamrnaases an aren eurumeween asore an souze s cromaoraroresses,e anaeso anr o rooc,oe–oe anoncneracons. er moecuarroeres, an coece resovease oner euon veoc, caneeecenvua comonens oe samesaraemeneo searaeraconseas.e euon veoc o sae.e acve comonen oe saonarrzaeses ae reenonme oeracon aneea anornoranc comoson, suc as sca,osre mono orarce so resn o oranc e moeases an aueous or oranc souon o n-co-vnenzen, or oeromers. cromaora maeerormenon-ar mor a mxure o aueous an oranc souons.e asrearaveuroses. e,n eevaeemeraures, anor anaca as we aeroceermsss c “arraancsearsoen ans v anr eon”z, “mraoecnr sonuceosns on voro”e, anes “raoseen o enx sereusesnocne on vro” o rreero a cneauoarn asna aers, ncucae sarsesma.es,n cen v curou sress,e amnncsusuees cuuurses n.omeo arca reacon mxure, aerocerms “ransaonn vvo”, “roen snessn vvo”, an “roen exressonn vvo” reero anaoesns caracerse as an enzmacroucon ooeesase on seuence o or ceuar an a nassuurear sansseman.sn onu vmvaon s, asnemma,ncaune,sunuas, n ooracmerea oor wno.e oransms, orans, uezeermns “ee croauresue ocroeen” or “eraeucroens” reero aoee moecuea mae nomeo anens, anra or meca sues.e casses oeraeucroensncueu are roenormone oes,mmunoencroens,uorescenroens, reorerroens, en an ssems,s moermernaen reeuroaeonns.,ranscronacors, reuaorroens, ssemsorene oeen-erremas “nerraameeu ecncoenen” or “e aemranoeu acce sneesu” reero a seuence o a nucec ac conann an ma conss ou are nomeo seuences enence oeeraeucroen.eraeucenes asm vecors, , m or crc. coen naura or snec vra or , suesaenrmces “ anarmaeceuca comoson” reerso a comoson o susances comrsn o acve armaceuca comeors suons manacese, sourcmu aase aasr am saoceu,cau, a accueeoause so suovoenn, asu aenouosr em caurrseorns., s cuosmenossono,nncue nsanouasr ncoes,meosoom ceosm, oorsonossomesan seusoerns exonr.eermarmaceuca anroensnn vro ann vvo seu. esson o aeraeucenes reescron oerawns reaeon coownnunceoan weescer aoenn oeera ewmnos.moernse oureonsvee ononus wreeer unersoo wen sownnerawns emomens, wc areresen e anenvenon,ere are owever,aenvenons nomeoerecse arxeme. soue unersoo, emomens sownnerawns. ranemen annsrumenaes oe -mureeu sanoowssneenera conce oerearaon o crc anaos comrsn -ner- es sexnemese usn -ner-a. rs,erecursor o crc anao aranscro zeurn ann vroranscron reacon.ueoeresence o ae′ ennnaor-ner-,eranscre conans a reacveunconarou eer enzm.aecx re,aceon′ e onr, rreeaceraveu,n cceomnacar roeaucosn.ncoreo′ra anen′ roeacveuos-sneca, reacveowars eac oer eer sonaneous orner nconarous are exame, arecursor o crc anao -ner esence o a seccrer. s an urn .ueoeresence o aranscronn-ao or -ner-a- s sneszeranscre conans an eeneamne more a-neer-′ e or -ner-a,e cemca moe ae′ en reamen weroae.s causen.erecursorsrs rose, wcsroneo nuceoc aac e moe.nas, seeecve oxaon oe′ aen.e.a3easo anrreverse covaennae o ’s′ an a′ enosn. o a reucve crcure s woawss us ae co asm aarrescounrso or eonrz cmemacca a cnrcu caermzaca crcuarzaon.′-moe re- oe same seuencere-crc w on.s a reerence,′-monoosorae caaze ase.e susr as use as a susraen enzmac crcuarzaon anaze ,: =aes: an,rourcs o cemca an enzmac reacons were eermne ensomer. reersonear eea,x .ees oe reacons wereen rannrom no n. n marer. reersonear o o aurene sarowrsec aunrase-meae remova onear neresence o crcuar .ames a mxure sor o crc anaore-crc anroucs o cemca crcuarzaon a seecv oere-crrocsesn aenar crc sece ws.ereerreaeas we arearomceenssvee sa′m exoensuc weearese anazaese reersonear,: oen = ran:,nromrea,x no. r ne.ersonear en marer. urse.a smowess a o aa csrec e asnaoon ascsarco con armnes cnrceuararreocuorsoo o cemca crcuarze -eenen enonuceoc ceavae caaze ase an rre-crc unerwenaren oonuceoe,e crc anaos nce.s suseuen .nresence o eecroorec mo o anearrecursor.ne oe a resu, nce crc exsowo moecues oower sze ran, ceavae oenearrecursoreas reersonear en mar.er. con reeorsnso:ne,: =:, rea,x. n. ar oen rannrom no amurees s oo aws aoenacr mamareer,e a eneecarrorecoress o crc –nuence oe crossnneree. o cemca crcuarzaona m ursor o crc anaore-crc , anroucs es o oacramees wxeurere usre-crc an crc were anaze .wo =:, rea,x an one weownerar cae: one w er crossnneree: .e mraon o crc s soweranraos osnnenarere.e: =:, rea,x areronouncene w aer crossnneree ecroorec mo erences rom no n. . reersonear oen rann reurceo sn oow ms annm oummrzeaeo enne o conormaon usn comemenar oonuceoes. recon oerar srr srucure o seuenceenerae w o we server. enerae wn′ ens aoucure o an seuence comrsnann reons o seuence sance′ a nmosnerer w aen r seearvcero.neewereencemon.sunorecsacee,ncea sec′rao o se-uence- ma coromee rmeceonraer a° or° eorewo or aerrzaon w one oe occurrence oe ooennuocmeoenonesaca-e.emcreeraseences oance nemw eemenss′o annan′ enns.caese crcure so wwass uesen asue ancreec our csoomrorem ceenmarca crcu oarzoanuocn.eoeoers on cemca crcuarzaon.re- was anneae w one oe comemenar oo ee oxaon se,erecursor e sames were anaze ,: n =uceo:,esrea-,x..ere reacon, sames orecursoreore reacon anroucs o r s a reerence, werencue. reersonear en marereacon wou comemenar wo rom no n.e reave amouns o crcuar.r reersonear oen rann susrae wereeermne ensomer. ouc,nearmer, an unreace u arne so-ws an ae.me searaon onear an crcuar usn- ,- crc was c same conann a mxure onear an crcuar re-crc + re-crc wraosm raesoorvaeen u ssamne croeneonerse.n m-oes an co cnoumonnss:.s a raecerernmcea,near nacaoumnm - m . - m soum ™. m . or mn,- n mn, or m roosae, m mnow. mmn a°.- n,- n mn, or ma ,. conons:arram- x. mm - con2 4a3. or mnow. mmn a° - ,ons:ee,re.-x. mnm mmnow -:. m mmn a, °.e,. - m rouruecs s oow csemecuar ccarcuonar oza croc usn reverse-ase-ressureu cromaora. conons°,exam n ore-crc were cromaorae unerenaurn asorn euae was coemcoenu amn ac seeaaeraeuenr aonr aacceoonnsrea m coxrruerse aosn ae mooeearsee.e- asorance.ex,eracons were concenrae an anaz neas o an aer crcuarzaon. –recursorre-cr e , aon w -uceore – concenrae racons, c ,r – crueroucs o crcuarzaon,- rannrom no n. –nea crononesn marer. reersonear oen ure crc snracon. : ,: =:, rea,x. neuarrere s-corwcsenu aennce c orc muoarease comoson on- o crc. mxure o cromaora waserormenresenccerc s was resove w - .ac reammonum aceae oerenon-arn aen:exammonum aceae ,-meexammonum acea-eee,x-amsomoronumam acmeoaneum aceaue.ammonum aceae rueraemen s.ows – anear anaesns o s meaerceer crc recursors an crc aer anase no n. conons: ,.: re =ers:o,nearrea,x oen. rannrom wure unm soowese.e.ran enszamoanac aacv o oan cerc crc anaocsrcn vro. or mo cees, w.eer.e craenmsecce oane crc crc w unnauranae, an crc w a ca srucure encoa aussaucerase, a reorerroen secree e cesoe meum.e n crc - were use as a reerences.e ce m crc recursorsre- ,, an oursosransecon an uea werearvese ameons,,,, wuceraseoumnescence assa.eeeceseumonrescueanncecroamo an omee roenosr werasr soumemnaervzees eorrorvearse reoraeseunm ane ssacnenacre. erreor oaaere mseenanene.raervesromreeneenen recaes, weruererans secowes wera unnsmaoonae acv o crc anaosn vroconnue. ces aussaucerase, a reorerroen scecrcre or moe crc crc , encon crc - were use as a reerence e cesoe meum.e crc recursorsre- ,, an oursosransece.one a cne u mea werearvese ameons,,,, wuceraseoumnreessceennc ae s aass seoruancaon oe reorerroeneves recaes, errorars re naa.r eerrora oarees meneaenne.raervesromreeneenen euare, mou sseowesnrecra cnessa aosn wae ac asv mac oro carceses aanaosesnro vmro.o, ,e,ransece w unmoecrc or moe crc crcne marrow en mconoces wereucerase, a reorerroen secree e cesoe meum.e crc orenaussa crc anre-crc as we as mocranseconwaernsea o wceursorsre- reerence.e mea werearvese ameons,,,,,, anreo uusers as aransecon or,,,, an oursosransecon asn case oenrc ces anos oruancaon oe reorerroeneves wuceraseoumnescence as useumnescencerom ameons was summarzeoveeoaumne sa.eeece normazesoe averaeumnescence oaneor scencea was suseuen ervesromreeneenen recaes, errorars rere csercn a san.are erroara oreese mneeanne.ra u wreere sranoswescee wransa unomnoa aceven ozm carcca a onaaonesn c vrcro.c,rc ,ea,e moe crc oanerom arecur , crc , c sor carrn -5- ae′ermnuscrc , arce′erm annu csrc or moe crc oanerom arecursor carrn -5-a roen secreecrec , cerc an crc enconaussaucerase, a reorer reerence.e cesoe meum.e crranseconwaernsea o was use as aransecon an ce us meeoa werearvese ameons,,,,,, an oursos assa.eeeceumrnesucaenncecaromon a omee reoonrser warso seumnmeavrezse wouvceeraeseoaouummnneesscceenncce aese wnaes snuseeureane nrorvmesarzoemroeenee avee e r nraeenu remnceasceesn,c eerro oraarnse reroersenn a .eaa e mean . sanar error oranusreece s wows moeraens carcona asc wv o c-rc5- aana,osan- vro acnonnaue-. a c′eersm wneures crec crc,r,ecur esnocrsonre-carucssauc,era,se, a re weorreer useroe asn s aec rereeerence.e ceesoe meum. arvese ameons,,,,,, an oursosranseco cne a mne ua were uancaon oe reorerroeneves wuceraseoumnescence seorneraervesromreeneenen recaes, erro assa.eaaresene . rars reresen a sanar error oe mean u wreerera snoswecsee wrans maoonae a ccrcv os o crcaner anaosn vro. , ,ea,e , ora- ae′ermnuscrc , cormcrecurso anrs carrn -5-a,a-ucerase, a reorerroen secree e cesoe me crc enconaussa was use as a reerence.e ce me um.e crranseconwaernsea o an oursosransecon an useora werearvese ameons,,,,,, oumnescence assa uancaon oe reorerroeneves wuceraseumnescence.ea.areeseeneceenumenesrcaenceerrovmes arommereoensn wease snumenma rrezecaoes,v eereoa reresen a sanar error oe mean . rorars u wreere sraonwsseceer wansa uonnmao acev an o c mroce a cnaosn vro. , ,ea,e -monoosae, --, --a,rac-s, o oraneromrecursors carrn crc , crc , cr5c , crc5an crc enaco-n aaussea′ucermenus a reorerroen secree e cesoe meum.e crranseconwa rase, use as a reerence.e ce mea werearvese ameon ernsea o was an useoruancaon oe reorerroene s,,,oursos-ransecon eeceumnescencerom a ves wuceraseoumnescence assa.e reseneneraervesrommereoennse waesn suenmm rearzceaeso errvoeraerso reareusemnn aes scaenncea.r ereaa e mean . ror o weruererans secowes werans-maeona asceuvour on cercm1u annamoosen vcrorcconnue-. m1or mo ces crc crc -m1enconaussaucerase, a reorerroen secree e e meum.e unmoe crc w urn cesoe crc wer ecrc an moe crc w urne , an e use as a reerence.e ce mea werearvese ameons,,,,,u oursosransecon an useoruancaon oe reorerroeneves w receercaasees, eorrourmnaressc reenrceese anssa a s.aneara erarorr oesenee meannera.ervesromreeneenen aure sowseransaona acves o crc anaosn ann vro ce-ree ssem.ex or moeec curoccesa ane w caasncuaeor w a mxure o7crcsunmoe crc wasmmeaeaceno a m-ceroenaenransaonnormsnuceoe.e reacon x was ae aneumnescence wea rsea meearner ,oe. o-coeeneraznen an mean an sanar error . sure a°.e assa was reeaeo cacuaee eaeescron crceresenrecnuvrseonr coonn raenaneswoo c rrecaucavreuncocnracrous a,n oanoe as oea′n-een an c arc suecarzaon o a en.. e reacveunconarou ae′-ensncororaeno urnon en azmea′c- snesoss-s.e.nne vcraor,ansc ereorn en rzeacmaonc. reaceon′-e onr, rreeaceraveu,nc coneamcraou reacsnocno.roraeno reacverous are reacveowars eac oer eer sonaneous orne e′ an′rer, suc ase aon o a cemca reaen, aon o a caass, a cresence o a secc resence o , or a canene oe souon, ec.eresenavane oemeraure,e rous reacveowars eac oerncue,u are nomeo: aears o′ an′ reacve aee, aze an ane, aze an ccoocneervave, amno ann caraee,raze an uorosuonrou, aze anosne, ec. oxcrou, amno an re.resenaevreec euxrasomrse a o mo aunec nouncaecro acu aae′-ense mo -ner- . mo cane reansaeurnen vro craannscerreare n vroranscron.e′- carre ouneresence o an -moeranscronnona reaconeranscronsseneca, as une oer meos o cemca crcuarzaonorseeaeor exames.s e ecenc o so-ase sness.e′- mo,e anrecuers nour’cseecns nome -ner- areone aner.enerse mae anear orrance ac com canna oon oe s ememeen cs,e sumcca as meoeecaesesuaces as or c aar-omeraeua onrosne can aenraso, o.re m carc conanrec muorsroer c coamex aona mocaons, suc aseecaeaes or-meuanosne ca anaos, as near o nonerere w e crcuarzaon reacon.sseneca, asn conrasoon e ase crcuarzaon,e users no resrce e acv an susrae-secc o aarcnzmac e cemca crcuarzaon reacons awo-serocess.rs,e crc uar enzme. oxze weroae ae′ enonrouce a reacveunconar recursors seecve o .s occurs va rn-oenn oxaon aneormaon oo au reaacavee ae ae′ en ervave oeermna′-en rose.urne secon se,e′-enacecceaerveae sonaneous reacs we′ mo.s se reures a reucve envronmenor omvae nereromrmeanacee. occu ars re, muacvne enev wronomeenrocses esnsrurereversuren.xces reaocrone,re reucon oeorme ′ reacverou ane′aee mo′ reacverou,e cemesceanc ce ore′- mo reuremens concernne cemca srucure ore seuen cuarzaonas no eneca, asn conraso crcuarzaonecnues ce oe crc recursor.ss anornao ssems,envene cemca crcuaase on acv o caac nucec acs consruc connorerearaon oe crcrzraecuornsoro.es no reure seuence enneern an c crrccuarzerecuarsoers. cosn aa cnomnarson, mreocurs woresreearrenar ae′ mon means o an suseuen seuence were sueceo enzmac crcuarzaon c oosae moe an anenca .nexece,ee o cemca crcuarzaon,aa onre cs eoemcaernsono. ease or aseesseenen onerecursor seuence ans oen m n.zmac crcuarzaon,s exoen cucrceuoarocooes oon ases carsc, enon anuacoeos oca asnseas, a cs wemec aas e cerccuroarzoareonc c manoe ver asseas u.sn exxoonnuucceeaosesc canesroons aess oans aow m ooneecuoessan cuosnanneraere′ osrom crcuar s, as crcuar s are ressano exonuceocross aseacrer′ en.ne oeran, rocessve′ exonucease,as mos common useo e3′ an′ ens.ases a crcuarzaon werereae w ase ..n conrr suc assa.eroucs o cemca were ressanoase ross. aroerecursors,e crc anaos seneconvuec aeoc assas arease on aenomenon o ncn annearzaon o crc.e- moecn seuence-eenen ceavae o a crcuar moecueeaso a snenear moecuue nes o o e - sumaa sezre u s.zen esx.e es.u25oce arsassaan e c,a c anea neva aree ero o nrm aene war oonua m cseoec,u ane eenaosnuocweaoseneaar ncs erno oucs oe c c eoes were useor ncn o nc rcemca a cnarcouasrezaasonoe.rn.earez aassoan. conrmea,n conrasoerecursors,27ecraonsoreoc mao, crc o nuce anc acesrs areeccure er sze, care, conormaon, anooo.26,eecrooressnenaurn conons.s.ors cane searaeurnoacramee marxs a crucaacorore searaon o aes crossnneree oeoacramee onear an crcuar usn a cross-neoe onerooo.ecroorec searaon reuar or commerca avaae aarosees.27acramees sncaneeran w casees c,r scuucar azsa seonue encecse cnocnasn anec ae′oe se seuueennccee, oe cerc curacrzaonreceurso cranae.nc.n some reec aursoron or a czoem anem aenecar neu ccoencor amca oon oomereraec.urso.r.ursnn aen r oeacoomne,r we or anoemas oeer an esn,e conormaona cane oerecursorecreasesesanceeweene′ ma ..n conseuence,e omasance an orenaon oe′ an′ ensn ancrea′ ens crcuarzaone .. seenceureessenevenovemneonn o as cor roemaaeosroa mec moes oosr oeanraeaorn an an crcsouaaron o r cesrocuon. anaos.s exrecvuosuosn c rreomoraeor maeons an o crom seaoura- o crcu.11a,r12z, 1a4ons mroeucos a weorwes oansee on sze- same comonensase onronamc raus ans consereorove oneo searae cromaora1c1, 12c, 1onons. - was useo resoveeroucs o oe m-asees cercwueaernz eaacon o.er, a4sn wees aes coasrmes, seerroecuurcsosrs aunc c arscreen sronns.cann case oer snn moecuar sze suc asosrene oroeneneoxeeronamc rausoes rausseecrceomers crcuarzaon.28sroer was useo searaenear an cccomers us ase uon cromaora .owever,eecreaseneoes raus o an moencuee cermeaon crcuarzaonas noeenocumene.ne cou secuaeanramoecuar ause s conrueoe exsence o aanscae o conormers, mamnsenuneracons,a cane oneerenaon ooes ra o crcuar annear .29s couen aces ooooca - uner m cononseasoe coeuon onac an nce crc. urnee12,o14exan w anonv-eexocmanen oenvenon, weounao sze-excuson cromaora - oe same moare mass.-. arensucen eecveor searannear an crcuaroee mos suae meoore seeavraersoen- o casrce - non-ar moe wasoun waserorme usnosren-vnenzen ua rreszansonroucs.,.romaora mxure o an aueous souon oonar reaen w ac, eevaeemeraure- °, an a onear an crcuar s aece comoson oeeo mnore as a moease.e searaon reaen.e mos eecve resouon was oane w me n-ease, namee cosenonar w anearraen o aceonre-,n mn,owrae.x mammmn aonum° aceae.., aeresennvenon aso reaesoeraeuc acaon o crc anaos.oemonsraee a reocarerro oen,roaeusns-eancuocenras ce,r wcere sn anaesozes anne enxeresesevern c,e csr.ceev aenas oose en recoorner raone un wereerne case o exresson o cemca moe crc anaoscrc ,ncreasnemouraeo cnr ocroecnr ecxr ..o moe an unmoe crcs exe ..s resu sowsaessonn comarsonoernearrecursorsre-crc - conex oeraeucroen e cxermeca mocaon wne crc anaossenecane aroaer rane o m seuencseson.oeeremonsraee aca oenvenon, crc , crcuar s moe ws w aas cenmvecsaanee.rce unmoe crcscrc , an w moea srucurecrc , crc , crcrc , c wrcereese a.n cersce va, eaureverse seuence eemens,ncunu nomeo a′- comrsn aarnans seuence o an aorac.oa, across ansances,roucs resunrom em crcuarzaon exe aumeneransaon ecenc, asusraen.. cemcanounevesae weereoson oener connecne′-ca srucureo m ′ enas an erennce c oneransaona acv o con crcs, crc anaos moe w one oree were snaessze an-r-a,a- , ana- an enconroenaussaucerase rooneroen exresasnosnecne comnaor ces. moe crcscrc ,, exe urermore,n vro exermenssonoernearrecursorsre-crc ,, .. comaraeor a o cemca soweae exressoneves oe reorerroen were resus sowa crc anaos m woe var corucs cem acnaoscrc ,, ..ese conex oeraeucroen exresson. a moe srucures areenecaneroansnavesaeenuence o aner connecne′-ca srucureoe m ′ en one reeonearen ac cvas o co-n crcs, crc anaos moe w an -5ner, one o enconroenaussaucera-sea wer,ea- , ana- , an unmoer crc an crc ,, exe erro senn exesrzeesso annranseceno ces. moe crcs moe ..ese resus sowa crceve an coamosar weo va urnomoe crc an srucures areenecane conex oeraeucroen exress us cemca moe eraeuc m.oweve1on. -meseuournemr,se anevoraaon mo o mc1a aonn oae rre nuocnecsanoen ucnaw nanuceeo reaascesoenc o oenaosrueuncona o eemens, suc asnernaosomenre carres oe auocaac seuences necessaror crcuarzaon meo,nucn aeraon r seconar srucure.n os, weese weer an enoccc ca srucure sner vae susueore eemen,ereacaneransaon o crcuar cou serve as a arorne m1ase mocaon.os en,enearrecursors were sneszecrc vroranscron neresence o m1-rosa an sueceo eer cemrouca onr enzm caecs. crceuoaurnzaoan.oown san1arroceures,e resun crcs wereranseceno comareos m1-re,e c crocunerar+,m as exusraee onn a m.ar.na resuconnransaona acv accornoeresennveno areers ncaeae crcs suc as m1. crcs comae w m o mocaons ansse osrsmereno w vaas coaneucee ca- coemenarennraen esxaon mecansm oe cae crc anaos, an crc n a ce-ree ssemra recuorcesseonsaevee,s o unmnoeeres cenrcce o a ca an- meoeeransaonnor, name m7s nuceoe.oa, an e nen concenraoneo a reuconneeve scaaonnenor crc , we no sncanmac was os oerv reeo ornereroraensna reosnu on crcromeran.sa.on oncaesae crcs accorncanosme.resennvenon, ar s roucon va a ca-eenen me eers crcsaeaoroen aes anerreescrons eascreo:ns o emaesnearzeasms an corresonn seuences w name oe a ennzmroee ursees.orounmeanrzeascorn oon:easm –esnaon o aasmesrcase – seuence encoene e asm seuence –esnaon o a reon maeor seuence eemens –′ an′ unransaeas′en an –o′aen,ro oene-corannscr seuence , – s meoueanrc eexonwcnosnre cam oe′ ′ orarcuar seuence.30:uman oecen cacuae nceaomocars vrus ,: eaon ′ or′ seuence, : e: enancereenuorescenroen re sseureanocre, snucc:aa vusrsusa-ura,uco: snec , nuceoe-onoaenne seuence. erase seuence,: asmesarcaseseuence eemens en-126-10aam seuence′-′ ′ancmmmme - e - - - - - - ... amar uc -x -x - - . - ... s o - - . ar x . s oza e -x .. cso sacer . e:s om uc - . a crec anes oer cemca o x - . crc –esnaon o r enzmac crcuarzaon.oumnescron: o an s a crc an corresonnre-crc seuence –esnaonransc euence encoene emaeor naor – comoun use as caarzeonnaornase r oeraconrcuasaerza uosneoer –e c orcu reaarczaononcemca o oron enuzcmac –normaon on weer a comemenar oonuceoe was u eoe crcuarzaon reaconrcuarzaone –ra seurnarcuar crc roucurn crcuarzaon reacon,ceeornm onee crc ore ccruurseor c crocnuvaerrzeaoonarrcuar anensomerc anass. oucs cccr rcuarzaon rcuarzaon rccrcseuence-nao5-rnz.eme .. oonuceoe e- ccr crcc - 5-nz. em.. - wo crrcc--aa55--nz.em.cc rcrc- 5-nz.. w e.woo-crc-a5-m.nz.em.. w woo c c rr cc- 5-nz.em.wwoo ccrc crrcc-a-55- n-nzz...em.wwoo ccrcannzz. .crrcc- 5-. . nz.em. wo - cccrcrrc- 5-c -5-nz.eemm. crc -a c a5- em crrcc --nz. . - c5em - ccrrrccc - 5-nz. - em. c ccrrrcc -c -a5-n -5-z. em. c crcrcc cr -a5-5-nz. . crc a emcrcc aa-- emcrnc a-a -em- exarcee:s o comemenara-oonuceoes useor crcuarzaon an seuermens.oumnescrons: –esnaon o a oonuceoe seuence oonnucceeo –ee.snaon o an seuence seuence – seuence oeesnae seuenceATCGCCCATATATTGTCTAGATTATCCCTAACATTTAA sAeTGuCeAnAcTeGAAAATGCTAGATTTTATTCCCTTTTTTTT GAAAAGGCCAAAAAATTGGTTCCTTAAGGAATTTTAATT TCT CCT CCT CTT TTT TTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGGGGGGGAGGGAGAG TTTTTTTTTTTTTTTTTGTTTAAACATTTAGGGAGAGGGGTA 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[0002] xames oeowonow enxam exames seso aure nroovee c sooneseoreem asonsmraene ornveenon ano exans asecs.envenon.xameemonsraese sness o r monsraneu scoe oe rou suaeore sness or eresenaveranscronnaor conann accornoe cemca c e-crcs – sarn maeraore crcuarzaon emonsr rcuarzaon meo woe cause as a reerence.xames– w anaeer soun seussa oe a roerreseen sanveesrsan oscr caoennrea-corrsc conasn –n sa cran sr mucauere eue crcuarzaon accornoe cemca crcuarzaon meoar raore xames–escreerearaon o crcs accornoeouces cae crc. crcs oane sae-o-e-ar meos us nvenon as we as reerence eroceuresor crc urca en comarave sues.xames–escre evauaon oerans on an srucure conrmaon.xame escree xames an aona acv o cae an unmoe crc-msn a ce-ree ssem. crcuarzaon.x escree omzaon o conormaonor oma cemca ces. ameescrese evauaon oeransaona acv o crc-msnvn xame.ness o -5-: e. men,eramne., mo,ma was aeo a souon o ane- - eser ammonum a.ceameo,m,an m, -ra.ee arn ac mn o vorexn a° aueous souon o mxure o ~.ex ec ac31, were aeo aor a co , a souon o3- mo,. m an a souon ou- mmoe wxas ae,, an me r weaecreo cnomne we mxure.era, soum ascorae m, souon oreammonum acea mexure wa,s sr,re a-°ra an monore .erwoours , m, an soum ascorae m, mo weree, a a soeu, aonn oe rueac-on was c comex overn.e reacon wasuence w a onnue - x mmow m n resove usn – coumn:emnm aceonre e mn a° soven: m4c . soven: or mnuon:romo n mn,enrom o n mn,en ree ree.ons euae conanneesrerouc wasreeze-re an resusenen waern was oaneon remeovoereraces o sovens an ammonum aceae.enarouc -5- w e.m o a so-u: monz o = anon.,a cra wcua aemmoornum aceae.- mmz:o, ur .ness o38581.. xame6163- m,-ar,on mm,azomeon.,. m mm.o,e re eacuvo.n w maxs auree waso s a souon o . an m2o60nore - rre a roomemeraure , ane-azoeam.nee.r mvmeoo,urs o re,ac eonu, excess wasrose w waer .e resun mxure was srre overn a rovo.m weams aeraeu,reo.owee . mmo, monore - .er comeon oener aacmen,e souon r weaac wonasroureess w was er noecorc ac a. un was reace, ane mxure was srre overn a roomem. areuraamse mxure wasure on -eaex coumn.e euae was concenraeoe mo, m.onum sa oenarouc as a mxure o′-′- reosomers m260, xame.ness o3-m7 soemersoe m. mmo, , euv. was aeo a susenson o3- mxure o a roomemeraure.260,e rea mco, mon . m, ane resun mxure was srre wasuence w waer mn anro erxesrs was monore - .erourourse reacon neurase wa anure ona ace w-eeaex e coerumnx. m.e aueousase was e areammonum3sa oenarouc as a mxure o′-e e′-uae was concenraeo m260, mo, . reosomers . xame.ness o3-m7mor3-a m3-m7,.m mx o, reomsoom2e1r wsere sus mene260,nmo., m m a,n. enuv an mm-,.mm . euv2.60was ae.e resun mxure was srre, ane reaconrore2ss w mo, .er comeon,e reacon as monore - e wa wasermnae e aon o aueous m, an we usse a ou aseneaorra wen so-a o a3.erouc wasure semrearave- mn.oeceracons were comne anc ceoonnren ammonum aceae .,. over mxure o′-′- reosomers mcen,raemoo r,emove wae excess o .erouc aerosaon.1z,2,2°60: =.s,,s om7Ga,nseom aesr a′ w,.eos,wer,m,7Gs,osmoemrer′ a′n.′,s.,m,,A,,som′e,r′ an′,.s,,3A,somer′ an′,.s,, ′,G.,3=.z,,′Asomsoemrer′,′ a.nm′,,.,,′,H-sHo =mer.′ azn,′,′m7Gsomer ′somerH-H′,.m,,m′7Gsomer′,G,.m,, ovemr7aGem7G.m,,′A,somer′ an′,.m, ′,′ wsomer′,,,,′′G,,so′mm7eGrs′o amner′′,,..–m.,,′m7Gsomer′,.–.m,, ,G′n′,s′G′o,m7G,me.r′m7–′Ga.nsom′e m,r ,.′, ,s,A,′m7G, 2-2,-′′m7G, s ,ssomer′ anm′,,.,–.′A,smom,er,′ an′G,′,′′G.,m′A,, ′ aA 3 m7Gomer′ an′,.m,,3,′--3,somer 7omer′ an′. xame.ness o -5-mmor -5-a me,.enermaom,nem.an,mo,ma. wase aeo a souon o ane- - eser.reammonu r mn o vorexn a° aueous souon o aor a mxurem o aceae-.ex,, a so muo,n o--mra7e an acec ac,m,o an w aer seo auoeo u- comex , m were com3ne wme mxure.er n o m, mo was ae, ane reacon a, soum ascorae erwoours a mxure was srre a° an monore . m souon ou- comex , m, an soum ascorae m, aon r weseorev ae uesn, ane – re caocuomn was connue overn.e reacon wasuence w a° soven: m cn:.e smonvenm:- x mmow mmn romo n mn4waeraceonre mxure:vv euon: conanneesrerouc, weansrroemeze-reo an resusnen men,n wenaernor mn. euae eraces o sovens an a ree reeonso remove o mmonum aceae.enarouc -5- was oanene erm o a souon o anonar w ammonum aceae.mo .. : mz = . ,. eor.--: mz, = u.r,- w2-: mz = rxoaman-e-o.ness o--er-uoxcaramo-e--′,″-meoxrenmeox- e a.r- °o.- r mm- .oreer-u--ar. me, noeucn. ar eaman m a mureas mxure was srreor anoer.en,e was eveao.rae m w,.o mmo was ae anesoae as cromaora scae coumn,– waern acueenoen anreeorouc was evaoraon m . mmo, o--er- ve aer meoxrenme uoxcaramo-e--′,″-1ox-roan--o as a weum. .m,,r,z,. -,°: =.m,,r,.3m,,r,.m,,r, ,3=.z,, -m,,.,sr,,,.m,,,.rm,,.,,,H,H. =m.,z,,o,c --oc,. m,H,H,,,.,.m,,,2oHc,H- =-.z,3H,H=3 D,.MTr.z,,,2′, =..z,,23H,H= =..z-,3H-,H=2.,.z, s,, m1322 EDzA,H,H H,Hz,,′z,. .,.3 B,oc.,.,.,.,.,.,-.,,.°,:. =,..,,..,m.,.,., rxamenem.eonxe-srso oan---o,′-ser-uoxcaroneeneamne--′,″-meox- -er- . mmo an ,.. mmon e ac -- m, overn eae. m ane mxure was srre a1o.evreou ace wras esvoaaoeraonas crom maora. m–m cnexane w. o uoxcaronez,eneam-n,e-°-′:,″ =-m.eomx,r,ern,me.oox, m,-roan- o-o as- a w,′-esoaemr-. .m,,r,.m,,r,.m,, - ,r,.m,,r, .s,,3 DMTr,.–.m,,×2,.s-,oc,,.3 tBu,s,.,s -,,,.3 tBmu,m,, ness ooc2-- -.a mnmoe o wer,e- aeo a so . . . m, m uon o-′-,′--er-uoxcaroneeneamne--′,″- wase sorrxer uneenr ar-moen aomxosroeraen-or-o.en m,e r.eac momno mxnur22. m.e mxure an. a m eac.e orancracon w re wasaroneeweenc vacuo.erouc3 aqasre overa24an concenraen o ove aer w eassoae as cromaora – cnexane w.vv ars oase vaoraon m31. mmo, oosorameoc2-- wo .s,,re,ome.rss a,s a, weoam. z, -,°: =.s,,, xame.o-ase sn,.s,, m ′--me- ess o--mnuceoe nr aceonr-eenz.o mae,n mosxnee w′--c.anoeno asceoornamree. m m a,nranmsoer wasssove srne conann so-suoreuanosne m ormeru reo a m moeraerevous w coroacec acnor, rromvaoae a anren vacuo.e mxure was sa22wase w aceonre, wase w aceonre. enor mn anene so suor wasere an erae w coroaeceose was oxze w m2nrnewaer: ann vacuo c acn22.e suor was wase w aceonre anre ..onc2aceon-r-e. m m an,ransmeorre waos asso mve srnnre c aocenoannrne so.- msu,o mxe w mo.e mxure was saenor mn anene so suor was remaceonre.eose was oxze w m nrnewa ere an wase w coroacec acn .e suor was wase2w a er: anerae w so-suorenuceo22e ceonre anren vacuo.en,e soro osoram was sueceoos aon usns-canoe-,- rouc was oxze we m,nrmonen aceonre. m w. . m.e ammonumroxe2 waer: an ceaverome so suor usn · n a me°a.mnee, cr:u vv. rous were remove usn cromaora on eaex eroucs weresoae on-excane raceeve aneoc-roecenuceoe was searaerommnuceoen aceercs a.ce aoc°rouosr we.re reemovreouc weaasnso aa seouono on-exroceacnee c saromaora on eaex anure ove aer evaoraon an ammonum sso ove-n-.m. m, m260nm,.mo,ur as a we so.e resue was - : =. mn mor ans eovmaeror aane.or mnesns wor esaono. 2tx6am40e12.183–-–:7.,2′-O,oun:. mer- : mz cac.or ness o m2--ma- -.- m, m.o were susenen,. mo an- . . an2a roomemeraureoras.erouc wassoae on mo mxure an srre eaex anure ove aer evaora -excane cromaora7,2′-Oon m . m, m2,.mo, as a woen s aon a.mmo-num s:a o = m.2mno-r-sooumne:r an.. monror60nsomsmoemr aenr-. : morz cac ss.oor385717285–t-–: . , xame.ness o-roar z--omorear.me ommerca avaae′-e m o .oa, - - m, mo wasssoven. aueous souon o a w. w souon oroarromenouene , euv. an an reacon was srre voro r. m as aaseranser caas were ae.e mxur us unu converson oe susrae on was oserve.e reacon was exer wacase exr macoree wmes w m m o: ovv m.xeur oer oanceaer e waesr anre w oaveerr a annero wuasera2aer4 a cnrom evaaoorraaeoorness ucnern reeuxcaene +re,ssure.evvro.ucna waes:ure as coumn xame.o-ase sness o-roar6mnuceoe m, mo. -6 snersosa orno sonu scueoore osorame aroac so -roar z--osoram m, mo, mooan, euv. an comoun nu e m, mo,. euv..nae oe xamceoee.ronuce:ss om-m-or,oar m6m. -r6 resunoa srouon n. m o .o ,,,′m-o,o eurv. anne m, mo, euv., m, mo, euv., e reacon mxure was srreor un3u m con,versonm oo,nu ecueov. wee wreas a oeservne.a oerner. souon oa ne m, m was aeorecaee-mazoe soum s, a e susenson wa4s cooenereezer a -⁰.erecae w a. coe mes anre2uner vacuum.roe as cenrue an wase w xame.ness o m2′-O, 7-arna m6amss oererouc was m. mm-o,-.r eouarv. a6nm n2. m, mo, euv. were a,e.e reacone mn mx2u2r′-eO,7was srre a reac°on a wnas mounenocree w a so.uoenr ou m comnverson o w-ma mzome waas ose3rve.. men,,e eeuva. oex resn.oe.rouc waqasuuerr.ee mnaeanrso oucon excmaon wea cs oromaanoera w one. xame-: m.z =nes.s, o ca mcu22′a-Oe, 7or36452-615244 m a m-z:.. m22′-O r,s7,- an mmxur, vv aonse mu e.:.er ca azener. m,mo,. euv. was ae, -ascorae. m,4mo, omex souon ,mo,. euv. an soum monore .er mnu eesuuv. c woenrveer asoen o.ee r seuacsorane m wxasur oe wsearsve sr are a° an wasuence w m . ,mo, euv..enaroucn wase reacon ou oe reacon mxurerearave- o en ammonum searae ..enarouc.mo was oane as a weower ae aceaeuer e. -: mz =., cacuaeor44662222-mz:..rosaon w xame.ness o m22′-O, 7 674m- m2′-O, r7s,- aner-ummxu,re.en azener. m,.mo,. euv. o was: ae, a-oanscsoreae. m m,u4mo, eu:v... come reeaxc soouon ,.mo,. euv. an soum .er mnuesu converson oe susrane w maxs ourese wrvaes s.rree r a° an monore m . ,mo, euv. anerou eacon wasuence w mxurerearav c was searae ou oe reacon racons conanneure-oc-roeceorouc weerernee azmmonum aceaeuer ..omnen mn o m an srr ere.e remannee wasssove ammo eor a⁰.en,e reacon mxure wasue w eum acne aamemounuemr acea.eou ae vroume. o. men aanroure onrearave- o mowz:er a..erosaon w e. -: mz =uc.,, camcuoae waso ora40ne56as20a w2442e- xame. emaerearaon oemeasms werereare conn a seuence o coceae, conann a esreraseroemmoeare ano a seor a secc resrcase ceavae,no ae vecor.o aore manuacurer’sroocorermo.eorasemass w seeereaneear.se w a resrcon enzme accorno x,am,e.eneraroocoorrearaon o -ner- re-crc ,-,,,, ranscro2nstock stomuer x xuockxfinor -m . em-nnearer- m-eanm. oasoec2. . . oonwrne oernesr: o waeer r,earcaon mxureseneae aove were mxen roomemeraurene ma an m snesrcmronuer x mrs- ., m2, m , ema ne,ermo,sermo, -ner-or -ner-a, mxure was veo,rexe,2,eno coecnrueerm, aon, annc omerase ermo.e reacon ermo was ae, ane mxure wasncuaeuaoero mrn a a°°..eex,arsoeuc. was,soae, rom wase r reesaocvoen m usxnure u-sno –na crocumn:o cemaneur - m, .,.esoae ~ mmn a° soven: m . soven: m x. a mcemonow. vv euon:rom o. n mn,enrom.o n mn,enreo: mn. euae conanneesre rouc was concen r aceae.,.,., ansoroan rae recaon: soum , ane souon was cooeor mn ao -.°- o.r ove wrnere a ae -°o.e eeuaeeen waas v coume = mn, ,°, wase w eano.u mc, cen-rue,- mn, enruen vacuo, an susenen waero aore ro oe cru,e°ro,urce. xame.eneraroocoorrearaon o st- ock stor ce k-crc f,,,,,, ranscro2nueruo inm x x x m . m . em2amen . oasoec . . . orern: wreaeern,sr oae reacon mxureseneae aove were mxe a °neoown a an mn sscrermonne,uer x mrs- ., m2, m , m ,oocermo, anermo,osmeraesremo,ermore.ncue raeaeco an m°xu,re wasemae, en2cenrue, anncuaeor a°.ex, vorexe, ane mxure wasncuaeo ase., ,ermo was ae, reacon r mn a°.e crue rouc wassoaerome .- mxure usnonarc ceanu , .esoae crue ~ , om.er o-mer one,e.r o x m rmeacoown m.x murem wna as res°ove so uvsenn- – coumn: .en sorovmen.:o m n mn.,e anceonreo:r mvvn. euon e:uraome conano: n. m en mn, rouc was concenrae recaon: soum a esre . were aeoe eua ceae.,.,., ansoroano.- or overn a -°.eeeen waas c veonurmue =e, m ann,e souo,n° was, c woaoseeor mn a -° . m, cenrue mn, ,°,ren vacuo, an s w eano rouc- ,- oe cruerouc. usenen waero aore xame.emca crcuarzaon o ,u,e so wuaso mn oxe werecursor - n , ~ ,re-crc ,-,,,, °o uer , m24, ma, . anncuae aemerarure. mne.r-er man, a reoomoe smoueroanur we,a asrreasns seorrueon on o seonc, were cooeo room was ae, ane reacon mxure wasmmeaeacena umeroae , m mn, ares souon o soum canoorore , mr wa anncuae a°.er connueore nex mn.e crue crc rouc s ae, anencuaon was onarc ceanu , .esoae was-soaerome reacon mxure usn usn- – coumn:omer - m ,. x o mmenoaw amoun was resove soven: m . soven: m . mmn a° o n mn,e n aceonre euon:rom conanneesre nroruocm was coonce n mn,en or mn. euae ., ansoroano.-. were anrae recaon: soum aceae.,., was cooeor mn a -° or ov eoe euaena voume =, ane souon °, wase w eano.e mrn, ce anr -u°e.eee was cenrue mn, , susenen waero aore crc rouc- mn,,- ,°,ren vacuo, an aonaurcaon an remova onear ,erouc wa oena amoun.or eowsee seconase eson. sreae w ase asescre xame.rcuarzaon o neresence o a comemenar oonuceoe ,u,e s,ou,on, o,e,rec,urso,r,-,-,n was, ~ ,re-crc ,-,,,, ma, ., an comemenar oonuceo mexe~ w,uer,., e, s m24, seuence anncuae a°or mn.era,eo souon wasr eeaeor cooeo roomemeraure.er- mn a ro anserre onenc, were , m was ae, ane re omemeraure, ares souon o soumeroae a°.er mn, ares acon mxure wasmmeaeacenear anncuae encuaon was con souon o soum canoorore , m was ae, an reacon mx nueore nex mn.e crue crc rouc wassoaerome - – cuoreu umsnn:onarc ceanu , .esoae was resove usn m .o somveenr:- mm ,. xn mm aceoonwr.e m emn a° soven: n mn,enrom o n mn uon:rom o eos ,en or mn. euae conannesre rouc was concenrae recaon: soum aceae.,., ., an mrona ano -°.- or. overn were a a -e°o.ee eueaee wnasa cen voruumee = m, ann,e sou,o°n w,a wsa csooee wor aor eean corc. mr,o ceuncru-e, m-n, oe,n°a, armeounn v.acuoor, a an sounsaenuercn waero remova onear ,erouc wasreae w ase asesc aon an eson. reeowsee seconase xame.nzmac crcuarzaon w ase ,ue wa sso muxoen o weureecrursor, m-rsn , ~ ,re-crc ,,,,,, or mn -, ma,. anncuae a° er-. menr aa ro,omeeom seorauuoren, wasranserarese onuenecr, were cooeo roomemeraure. , m , ., , , x, ,, m.rs-, m o2ocasenor., ,er , m, , ae, ane reacon mxure wasncuamo, an ase , , weresoaerome reacon m e a°or mn.e crue crc rouc was - oena amounxu wreas us rnonarc ceanu , .esoae . x mmow. mmn a°es soovvee usn- – coumn: erem , m n aceonre enuo:n:ro mm o.nn m aceonre soven: n mn,en or mn. euae conanneesren,enroromuco concenrae recaon: soum aceae.,.,., anso was aeoe euaena voume =, ane souon roano.-. were a -°.eee was cenrue m was cooeor mn a -° or overn cenrue mn, ,°,renn, vacuo,,°, wase w eano. m, rouc-,- oe cruerouc an susenen waero aore crc erouc wasreae w ase asescr.eor aeowonseae seucrocnaona asen remoevsaon o.near , xame.nzmac crcuarzaon w ase ,ue, so,uon w oas mexerec wursor comeme-nar ononuc, ~ ,re-crc ,,,,,, or seuence anonocmuaemee araur°e.orer m-n. eoe~, ,. e, seeae were cooeo r mner a raoo,meemoe sroauureo,n wasransaesrere ouneern xc, ,,, moros-c,ase mnor2,. m, ,, mermo,, an., , , , were ae, ane reacon mxure wasncuae a° ase., crc rouc wassoaerome reacon mxur or mn.e crue .esoae - oena amoune us wnas resoonarc ceanu , omer -m ,. x mmow. mmn a°ve so uvsenn:- m – coumn r.o soven: m n aceonre euon:rom o n mn,en wm o n mn,en or mn. euae conanneesre rouc waesre co anceenraoee euareecnaaon v:o soumuem = acea,e.,., ., ansoroano.-. overn a -°. ane souon was cooeor mn a -° or m, cen eee was cenrue mn, ,°, wase w eano. roucr-ue,- mn, oen,a° am,oruen.n voarc auo, anona susurenceaon waero aore crc erouc wasreae w ase asescreeowsee seconnas aen remeosvaon o.near , xame.- o crc manuacure searamonasz wuere-erorm-e se ornes mouar,ow-ressureraen aaraus eecor anuorescenceeecor.e mo wea ase wearms coosmaeo coumnoer or coumn oven, -rae aceonre..a se o non-auocave waer, examne oem er, uareamneoura,ma, -rae n- comoson, concenra,on an an we-reraeereac wa ac.ecm acer. an.aer.ovens oesreoner,eros ome.romaorams sorenear a°or nm were recore weeecon o asorance a nmre , n-manw., asorance secrum- nm, anuorescence oneres – xame.ase eson seoru monn o , wasncuae a°or mn an ra cooen ancea. ,o aoc-°,aseanseorue.r x , mrs-, , m2, ., ae.erm men r oeacncouna moxnu a , ,ermo, anase , , were orsoaero re us°neonraorcucs o ceeasnuon were ana,ze w. rec xame.ase assa souon. conann, c arncaseu ner, x m oonrusc-eoe or n, , m ,ermo waseae , m , m ex,2oocase o°or mn anraua cooeo°or mn. ae.er mn onncuaoorn a.°,ero,ucesrm oo, aenson waseererec, anaz,e wermo were. xame.ransaonn onn ssem mexxurea ec,uocecsa me, an,romea w.a msu, aen wn souon conann amno ac n mxe w m7s –2was aecuoaeor o ae°sa.e mexx,ure oncuaeor a°.ex,e reacon wasmmeaeaceno a mcr an ,oe. o-coeenerazanecuxazene mxean an wa ss aanear a en oae reaerner a°.e assa was reeaeo c rroreumn.escence was measure xame. roe sness n veror-anscr-on o ro seeu weansce s wnasesezreorm acecornneoreseencrevous reorerooco.31rs e cruerouc o wassoae onarc e o3-ranscronnaor. an′- uaaen.e crueaenrouc wa ceanu an sueceo′- onarc ceanu an suse ssoaeromeaen reacon mxure eesrerouc was conce uen resove w .e euae conann e- - ronera soeuon.recaonren vacuo an susenen waero aor xame. exermens .e,na- voum-e o oro aeo srou aonna warsoeu coenc wenrauoner m24, ma, uorescence cuvee x x mm.o o n anranserreo auarz , ,- was ae.e mna, a souon o a comemenar oonuceoe cooeo° or°n- mn.xmure waseaeromo °n mn anraua secrouoromee sson secra were recore on aarcse en, excaorn aen nm,, e emusseon w- a x nemno,na mmm, s sn.e ceeer accessorsvx, xame.e cuure anransecons moeus aenaenssr wacs ceerso armne mancrooura cees enseas,.es. ,e, , anea, as we asnransecon,x3ces were seeeer we o ae-rwomeoanee. marero nwex moano,c ceess. wenreeraaneore w .n eac cene, eac varan wasransecenrcae. sece w oecamneessener nvroen, ransecon waserorme manuacurer’snsrucons.urnr aam, , , accornoe acco ansecon,e moar concenraon o eac wasaenno ,un s ooao aenc eamuane am woausnr osue moecxuesn was a oeo e-ac we.re,,roarn eacsa rnea,con, n m oeum anncuae a roomemeraureor mnues.n anoerue,e wasue mnues a room-eme mraeureumx.ex,o x anx were mxe anncuaeor o a-weae conann.rerwar, oe mxure wasranserreoe arorae we sanar cuure c evous seee ces.ereransecon,e ces werencuaen meumrom eacon weon wsas w°,ra, sauranum. aroraemeons, u o meum was comee reace wwneoese amoun orouceroen, ane remann was anaze usneuc sres one.e amoun oucerase reeasenoe meum accornoe manuacurer’snsrsaano ecnooes,orman,, reaens, easuremens were mae usn annusconons.aeu remaneersceenrcen wamse rre,aana wm,e, o,aue.aes. eerences aa.r mene,.-aesraean,er.ae,uenc,s:..owe:ernu,. a.n vaensr,sa.e-oaoo,s-o. com-ewnaa,cse.ases,..naenr,an.sucu,on. an e anows,.nrzeewsa,.eeac o m enneern:ccneesuse oon-e- ::. arczcec.esseoecua-rera -uceccs ,,-. :..omersn.ore.o.e oanecaurnaaerauw,ea.s o.o-ousz,: e.,: raau.s,ozn,..-e..e,:wa.s a,nnrmwas. o mousee,ec.a.oaeruvree,ev.ewes accceecss,e..--aress,:.:..eecn an caracer-.:..ce... ,onna,a,-...:u. n. acccessezn- cr-cuar::s..aureoecnoo cvaenmovcsaaa,ao.n..ucasesecur,men.ea,aro.va,e.ren.ovao,nu.ceo.er cerscuaaar,za.on.ermnease-aare,ce. arucoccmceess ommaosee una-menos auonc,e.ucee.ome e,caeorcovas, .ssuae ,,o.sne .:a.rc ,eo mcuaran-, ,s,m. . a.n.,r:esm.a-zaa,o.n.. o:aear.oevaac,on.nar ..o.nan.oonesn. sneszeenca, c.orncs.uareza,an,s w.,.os-ora,m.aenaomeso enea,e. r.omn:z cu,r.ce.ranse,ao.n.emem.caca o monomercnear .ronoro s,ness o, annecous vro:nass oenecv en,.-.-a.rnow,.naon oroenness-e.uar:ocsra:nso.or. - rcuar s.cence , , aonaaraus on e,...a -. :. scence. acccesse auaocccaucmeasasre,c.ranasc-re,.. ecen exresson o crcuar aamersn ces usn mre-rs,..au:re:aso.ecnoo , ,-.:. s --- .n sewu,. c.rcuwaornz,e.. es a.a.o,xe.nsrvnneurarn,o vr.roaoanra a,onr.:onre v.v,o.aroamen,ra.nsauoanr voa, er,veor esr-, som. a.rreosu,:.n.rnone osnsa osa. ce.rc.u.arrocaececcnensseasc oerae a-na-oenaas u:scan:em. o ccceanscee rsozm,es sae-reasne saesccaco oeeoesns, ,en. . eu. .ar-ooow w-cas,..nerson,..nneern crcuar oroen an a cse,s:..:a.ur.eareormmuncaons , .:. s -- oecu:rcuarzaonmnsesmmunoenc-a aen,.a.nuxanen,.ransara,.nerson,.. ,ar:.e.amaa,,.u,-.e.:..moce..s.aonaccucreasseonn-vo-. reac.oun,r:o.u.c,n. crceucnca,rraa, .en,anc. ac.c ceaessaeua enan ,r m, .o.-esmacnnan ureuaeac-scn u,.-.emu:c,a:.om.muncaons , , - rcu o.ear. va:ccn.es aa.ns -o- an emern varaanns,..ean,,.an,,. e a-. so:cos,..aor,ce..c.en.os,acc.ces.se -as-es.ur:ren:.roocosnoecu auom.oarnn,,..en,,...au-e.m..,sasc:cceoss.eor. . .m s.ar: eers: r,.. ase:usrae canen reuremens. ua:rau., -, -. :. - -acccesse -- carcccuceasarsme eaxnoans,.. an-,uc-een,.rouermuenron-exon seuences se-sceorouce .eco:c,s:..e.searacn,.-., m ca,n:-o. :. nar.. ouce .srcovcc,s.evsearc , , -.:. naorcwauncacocncse asnse acaons.. connnreo oene exnressuorn,..eonenneerr,s.anaron,.a an ca‐nnroensne ,uswcrna.acccesse .ccena,r..evewas:er,..,re,.,.-ar,..-.: mua oooce, ,-an.o.n.c.ao u:rrascn.s,a.on..mnoecae′-.on- ane.o.uaoncaocsc ancneesseaern oanvu-eaa-rowac m.:o:ecs.uaerrveseoo, aanrm cans,r,ans.ae,roan, or w. s-, so .nr.sa .e-m, ow:o. aecs.ueun rsesso,u.on-nase-vvec ce,-s....auuraenruncuraenaomeccusa or an.aosen-o-u-m. as.oa,c.senmeurr,en.emcasorns o,osae,o.caon: moeuaenesrsro, ane.n. m exeraersmsaononsnn, sav.uns ou ceacse.a,ursc.ercaan aascccrzsa re, n.uceoows,en. euowaarsoac, m.eme′ c,a.raensssameaonnn,,...-o..ecueean,,..acemsoene,r,... een a,..ruecnuraeersee eanr-ecmaenas,,rv.e eax,nen,sv.-e cnrc.uera,r.. aececnce,esss.e ar e-ar.can,,.. eea, an.,-x.e,nsanv,er.an,saao,on-. o conr.ceu,ar..u,s:.rve.ann,..m-omnce,e..aea.nnos,.ne.. e.e-meann,.en,.an,..ervas:ve.ranscar.on o. crcauccacresses-- ::.an, es,.:.a:uree.no,m.munncaons , .:. s --rven-a sccocresse-e-n-uman: can:cer..oraesc,ua.r-,.an.cearven,,,..oan.o,,..:ran.s.aouosnss aen,u.n-caon-na, ro.-es oaarcm cccrec-susueasrn,.s- acazea,re,ne..rcu:ar.,emseorransacaccoenss.enerna-on-aourn:a: o.oecuarcences, rucura anz-aaesc,an.srcancesacuor-ese oa,ra.ernaneze-menams.orro,.mare,..nssno reavneswa.ona arnuess,o.enaacerora,ov.e momo,-.nerna osroomneersnrncerooo-ea,e, n., .neeer,. sn. c.:r-cum.aar-a,.ome -,oarc.cne c ecneas srasneecneas,r-.oeaacs nceec,ersos.eera u.cone.n-,ev a-e.-ome ur.e:ne. on:o eecr,n.me o.oe.nc,ne.san.a,n,.,:.. rucuorewo on,uma.naaasmeaov, com..exe wran-o a,n.:e:rre.,.r.:nrsouc,no..an rev,e. oranscarcuoens,..-o.ecuusasr cne,.,.se o, eec-ro.orec mo oeermnee seconar srucursree a.s an, smn.aara. arn.senseaccces.seucec--cs:es:ear.cern,,.aom,asu-e-rmosa.,..: meornecnueesz.,.ucerncmeze,rc,..sa.rer esae,a ar..eecrnronoerze-ces moosa,. o. suceraceoreer,, c.aenaarene azn-onoese,.-. e,...esseoe,.c.en,, , e-e. :. naru acccessen. aaro.smeocee. ee.cro.oreasccsc.esseoecu.a-rnereson,..,an,,..rc-uar.e m.raon: o-mears.ue,au.re.emrasrson,.,.,e s-ne.-ss,:ro:e.r:es.s ano-en-a a-caaccocness o ccc :cen: :s,:..e,..ous onowonana aou srucur se - eaonea,.c.a.em oocaecn:c aens onne, oonuc.eo:e.roerensa cs. acec.cesrsoeceen-s o- ,su_,-.:. narmacccessacuaor.uceccsesearc ervaavmeos e,.cenors rea,c. w. oexerszea,.e′ enes,.rovownas aeac,ce.seme,.. eneamne :ro.esonra ernzamac aascscacess asnen vvoran.saon.uceccsseose maorcno an,ua,a ee-ee.

Claims

ams . crc anao accornoormuaw n aser n aenn: eacne oer wnsn a veaeueromo an eac onsnenen ,3,2, or an eac onsneeenneen or an eac onsneennen,, or o , an′ a enac ea oc onsnnseneneeen,,r a,-a oraoen -mse aneuaarno osrnera cnac serucnn uree er.,n we ar neanu sraa, monere o cro umnnrasuersa a nu ncaeuoras,e moasee or unnaura .ormua: crc anao accorno cam, werens a srucure accornoormua orw easere-mn:euanne, naura or unnaura-meuanneervave or anao, qc1, oq2,1rosune3ensneneen,en, o,r or, weren1 +2 +3s eua or qq13Lasnq3sneenen,,3,aoen or aoen Ls a,q2,,2,aoen2or2aoen2Lss a, s snn ,ee c c -eemm e,ccaa -aoonn ,,,, -2s,u or, s a, uneea2r or anear orrancener a orra.ncener . crc anao accorno cam, werens a srucure accornoormuaw eacsere o-mn:erouuanne,s nanureae onr uennnaur,a o-rm,e wereuannne +er +vavse e oru aanao or, q1, aq2,1q3sne3enen,3, or1 2 3q1nq2sneenen,,2,aoen2or aoenq3Lss a, sn,e c,em2c,aoano,en,2or,,ao oren anear orrancene anL1anL2L2areneenen,,-e,2-a,-susuear, weren aeas one oL1L3ansLn4o areneenen sne cemcaons,2 or anear orrancener. . crc anao accorno cam, werens a srucure seece amon ooownormuas:. crc anao accorno cam, werens a srucure seece amon ooownormuas:a.eecae crace. anao accorno cam, werenener comrses aonaa. crc m aneaoo orecu srsnoresosrm oua crc anaosenen cams-, caracerze aw n =eren: a an- n e eaacc o onnssnneeeennen ,3,2, or an eac onsneeen or an eac ons nen, or an eacnneenen, or ,′ an ea oc osnnseenen,, a,-a oraoen s anear orrancneeenneer,n we ar neanu sraa, moe or unnaura nuceosease -meuanosne ca srucure ner comrses a naura, moe or unnaurasrs oxzeurn se oe meo,en aaeeervaveormua, wern =e a an-n: n eac onnsneenen ,3,2, or a ann e e eaa acc c o o onnss snn nee eeenn eneenn en, or or an eac onsneen , or ,′ an eac onsneeennen, a na, aura,, mo-ae o orr uanonaeunra nuceosease-ms anear orrancener, weren saner comrses a naura, moe or unnaura w crcces nu aea nxnos aon.cnue caae srnuc reuruecve envronmenurn se oe meo,eneesre re.acor. meo, accorno cam, caracerze a se an se are carre oun one .resence o meao4,, acrceoerrano a c aa cmonce,n craaraocnerezeow ma s,e a ae omeera muereoeosw carr°e. oune u.ere so mueoon,, arecceorran ao cam–,,n carerascenecrez oeaa3seeow oe m me.os carre oun car.re ou mneroese,n acceco or an nauora c,a mmo, cea orarc uenrnazeura nucaec s aec o oro smeera os coem meemoena arreo a recursor seuence. rea.con m mxeureo, a acconrownn moe caom o, caracsoerazeon, suca asere ccrcaon anna aocosos,o sazee exrcomue cromaora,e eecrooress, or-erormanceu cromaora . son w.cncseu oes au crcs no anmaoe aocc aorrncao r cams–nroenrouconn ann vro seu, cuures, anssue cuures. eacon mxure, ceuarsaes, nucearsaes, ce ar.cuar corrcroen anaro accorno cams–or useneraeuc oranosc meo,n oransms, orans, ceuaor aunconssune arnansn vanvos. seu, wcncuesus nomeo woe . armaceuca comoson comrsn o a crc anao accorno cams–.

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