iv. javaxiSvilis saxelobis Tbilisis saxelmwifo universiteti

zust da sabunebismetyvelo mecnierebaTa fakulteti fizikis mimarTuleba

Tamar farcxalaZe

pH-is gavlena dnm-is struqturul labilobaze da misi roli cilis mier dnm-is ubnebis amocnobaSi

s a d o q t o r o d i s e r t a c i a

programis xelmZRvaneli: Tsu asocirebuli profesori, fiz. maT. mecn. doqtori Tamaz mZinaraSvili

samecniero xelmZRvanelebi: Tsu asocirebuli profesori, fiz. maT. mecn. doqtori Tamaz mZinaraSvili Tsu asist. Pprofesori, biologiis mecn. doqtori mariam xvedeliZe

Tbilisi

2010 weli

1

Sinaarsi

gv

Sesavali

I. Tavi. literaturis mimoxilva

1.1. dnm-is struqtura da genis funqcionirebis molekuluri meqanizmi

1.2. dnm-is ormag-spiraluri struqturis mastabilizirebeli Zalebi

1.3. dnm-is spiral-gorgaluri gadasvla

1.4. dnm-is ormagi spiralis protonizacia/deprotonizacia

1.5. specifiuri da araspecifiuri urTierTqmedebis saxeebi (tipebi)

dnm-is molekulasTan

II. Tavi. eqsperimentuli nawili

2.1. sakvlevi masalebi

2.2. naSromSi gamoyenebuli meTodebis aRwera

2.2.1. centrifugirebis da speqtrofotometruli meTodebi

2.2.2. diferencialuri mikrokalorimetruli meTodi

2.2.3. mikrokalorimetruli mrudebis damuSaveba

2.3. miRebuli eqsperimentaluri Sedegebi

2.4. eqsperimentuli Sedegebis ganxilva

daskvnebi

gamoyenebuli literatura

2

5

14

17

21

24

29

30

31

35

38

54

59

61

2

Sesavali

problemis aqtualoba. molekuluri biologiis erT-erT

umniSvnelovanes amocanas warmoadgens _ cila-dnm-is urTierTqmedebis Seswavla. gansakuTrebiT did yuradRebas ipyrobs cilebis mier dnm-is specifiuri nukleotiduri Tanamimdevrobebis amocnobis meqanizmis dadgena. es fundamenturi problemaa, romelic mniSvnelovan rols TamaSobs iseT sasicocxlod mniSvnelovan ujredul procesebSi, rogorebicaa dnm-is transkrifcia, replikacia, dnm-is reparacia da rekombinacia. cnobilia, rom mravali daavadebis safuZvels warmoadgens defeqtebi dnm-is amomcnob cilebSi an mutaciebi, romelTac SeiZleba adgili hqondes genis promotorul ubanze, saidanac xorcieldeba transkrifciis inicireba, ris Sedegadac arasworad warimarTeba genuri eqspresia ujredis birTvSi.

msoflios mraval laboratoriul centrSi wlebis manZilze mimdinareobda kvlevebi, romlebic dakavSirebuli iyo dnm-is molekulis fiziko-qimiuri Tvisebebis srulyofilad SeswavlasTan da es kvlevebi dResac grZeldeba. am molekulis Sesaxeb dagrovda mniSvnelovani informacia, Tumca jer kidev gasarkvevia im meqanizmebis buneba, romliTac dnm-is molekula funqcionirebs ujredis birTvSi. bolomde Seswavlili ar aris aseve, molekulur doneze Tu rogor xdeba birTvSi dnm-is genetikuri informaciis Caketva/Senaxva da saWiroebis SemTxvevaSi rogor xorcieldeba dnm-is Sesabamisi monakveTidan informaciis transkrifcia, rac metad aqtualur sakiTxs warmoadgens Tanamedrove genuri inJineriis da samedicino genetikis sferoSi momuSave mecnierTaTvis. amitomac, mniSvnelovania dadgenil iyos zusti molekuluri meqanizmebi, romliTac xorcieldeba genis uSecdomo gaxsna/daxurva. literaturuli monacemebidan cnobilia, rom dnm-is im ubnebs, saidanac unda ganxorcieldes transkrifciis an replikaciis procesi, win uswrebs fuZe wyvilebis specifiuri Tanamimdevrobebi (promotoruli ubani), romelTa amocnobac xorcieldeba garkveuli tipis cilebis mier. aRniSnuli Tanamimdevrobebi warmoadgenen dnm-is ormagi spiralis gancalkevebis iniciaciis adgils. rogorc wesi, aseTi Tanamimdevrobebi gamoirCevian AT-wyvilebis maRali SemcvelobiT (TATA-

box - transkrifciis SemTxvevaSi, 13-fuZe wyvilis Semcveli ubani - replikaciis SemTxvevaSi). es faqti SesaZlebelia aixsnas imiT, rom Sesabamisi helikazuri aqtivobis mqone fermentis mier dnm-is ormagi spiralis gaswvriv pirveladi destabilizaciis adgils unda warmoadgendes energetikulad SedarebiT dabali stabilobis ubnebi (AT-wyvilebiT mdidari ubani), sadac moxdeba jaWvebis gancalkeveba, cila-dnm-is Ria kompleqsis warmoqmna da amgvarad cilis mier am ubnis amocnoba.

dReisaTvis bolomde ar aris cnobili CamonaTvali im Zalebisa, romlebic monawileoben cila-dnm-is specifiuri kompleqsis warmoqmnaSi, aseve ucnobia molekulur doneze Tu ra urTierTqmedebebi ganapirobebs dnm-is ubnebis despiralizacias da ra roli SeiZleba ekisrebodes dnm-Tan kompleqsSi myof konkretul cilas am procesSi. ucnobia aseve zusti molekuluri meqanizmebi am ubnebidan informaciis “wakiTxvisa”. Tumca literaturul monacemebSi gamoTqmulia garkveuli mosazrebebi TiToeul problemasTan dakavSirebiT, magram rogorc avRniSneT utyuari pasuxi am kiTxvebze jer kidev ar arsebobs.

Tanamedrove medicinis da farmakologiisaTvis erT-erT aqtualur

3

sakiTxs warmoadgens genis (dnm) gadamtani, usafrTxo da efeqturi polimeruli nanonawilakebis Seqmna, romelTa saSualebiT SesaZlebeli gaxdeba ganxorcieldes cocxal organizmSi daniSnulebis adgilamde (samizne ujredebamde) saWiro genuri masalis dauzianeblad gadatana da mis ujredSi Setana. am SemTxvevisTvis SesaZlebelia gamoyenebuli iyos iseTi nanonawilakebi, romlebic dnm-is genur monakveTTan warmoqmnis araspecifiur urTierTqmedebas (magaliTad eleqtrostatikuri saxis) da rac mTavaria am kompleqss eqneba unari SeaRwios ujredis SigniT, sadac ujredis fermentuli sistemis mier moxdeba mxolod nanonawilakebis daSla.

kvlevis mizani. Cveni kvlevebis mizans warmoadgenda: dnm-is struqturuli SesaZleblobebis kvleva sxvadasxva mJavianobis (pH 2.6-10.0) garemo pirobebSi; mudmivi ionuri Zalis pirobebSi gansazRvruli yofiliyo dnm-is Termodinamikuri parametrebis mniSvnelobebi pH-is farTe intervalSi, romlebic damokidebulia dnm-is struqturaze; dadgena mJavianobiT gamowveuli dnm-is struqturuli cvlilebebis Seqcevadobisa, vTvliT ra rom Seqcevadobis unari uSualod kavSirSia dnm-is funqcionirebasTan birTvSi, miRebuli eqsperimentuli Sedegebis safuZvelze Camoyalibdes mosazrebani (modeli) cilis mier dnm-is ubnebis amocnobis molekuluri meqanizmebis Sesaxeb.

aseve, kvlevis mizans warmoadgenda dnm-Tan araspecifiuri urTierTqmedebis saSualebiT ori tipis nanonawilakis (Np-PLGA da cNp- xitosan SaliTani PLGA) dakavSirebis SesaZleblobis kvleva da warmoqmnil kompleqsSi dnm/nanonawilakis maqsimaluri Tanafardobis gansazRvra, vinaidan arsebiTad mniSvnelovania nawilakma moaxerxos mTliani genis gadatana organizmSi daniSnulebis adgilamde (samizne ujredebamde).

mecnieruli siaxle. Catarebuli eqsperimentuli kvlevebis

safuZvelze miRebulia Sedegebi, romlis safuZvelzec yalibdeba principulad axali xedva cila-dnm-is specifiuri uTierTqmedebis meqanizmis Sesaxeb. xazi unda gaesvas im garemoebas, rom mJave are unda TamaSobdes gadamwyvet rols cilis mier dnm-is garkveuli ubnebis amocnobis procesSi, rac damyarebulia Cvens mier miRebuli eqsperimentuli Sedegebis analizze, romelTa mixedviTac, Tu dnm-is ubani moxvedeba mJave garemoSi, xdeba am ubnis stabilobis mniSvnelovani Semcireba, rac gamoiwvevs ubnis despiralizacias (gaxsnas). amgvarad, dnm-is amomcnobi cilis mier xelmisawvdomi xdeba fuZe wyvilSi is ubnebi, romlebic manamde wyalbadur kavSirSi monawileobdnen da SesaZlebeli xdeba cilis mierTeba am gaxsnil jaWvebTan.

cilis mier dnm-is specifiuri ubnebis amocnobis aseTi meqanizmi sruliad axalia da migvaCnia, rom sruliad logikuri, rac jamSi emijneba literaturaSi aqamde arsebul Sexedulebebs. rac Seexeba dnm-is amomcnobi cilis struqturis Taviseburebebs, Cveni azriT logikuri iqneboda, rom cilebi Tavis struqturaSi Seicavdnen mJava aminomJavebiT koncentrirebul ubnebs, rasac adasturebs literaturaSi arsebuli monacemebi aseTi cilebis Sesaxeb. es ukanaskneli kidev ufro aZliereben Cvens mier Camoyalibebul mosazrebebs cilis mier dnm-is ubnebis amocnobis meqanizmebis Sesaxeb.

eqsperimentuli kvlevebiT naCvenebia xitosan-SaliTiani PLGA

nanonawilakis (cNp) dnm-Tan dakavSirebis SesaZlebloba da gansazRvrulia is maqsimaluri raodenoba genuri masalisa, romelic SesaZlebelia

4

adsorbirebul iqnas gadamtani nawilakis zedapirze. sapirispiro Sedegia miRebuli uSaliTo PLGA nanonawilakis (Np) SemTxvevaSi, sadac dnm-Tan kompleqsis warmoqmna ar dafiqsirda. aRniSnuli kvlevebi miuTiTebs xitosan-SaliTiani PLGA-nanonawilakis, rogorc organizmSi genis gadamtani saSualebis gamoyenebis efeqturobaze, gansxvavebiT sufTa saxis PLGA nanonawilakisagan.

naSromis praqtikuli mniSvneloba. miRebuli Sedegebi, romlebic

exeba molekuluri biologiisa da farmakologiis umniSvnelovanes problemebs, kerZod, cila-dnm-is specifiuri urTierTqmedebis da genis gadamtani PLGA-nanonawilakebis da dnm-is araspecifiuri urTierTqmedebis meqanizmis dadgenas, mniSvnelovnad Seuwyobs xels gadaiWras mravali problema, romelic arsebobs, rogorc janmrTelobis dacvis sferoSi, aseve bioteqnologiaSi, ekologiaSi, soflismeurneobaSi da sxva momijnave sferoebSi, rac naSromis praqtikul mniSvnelobis Sesaxeb yvelanair kiTxvebs xsnis.

5

I. Tavi.

literaturis mimoxilva

1.1. dnm-is struqtura da genis funqcionirebis

molekuluri meqanizmi

dnm cocxal organizmSi arsebuli erT-erTi umniSvnelovanesi

makromolekulaa, romlis funqcias genetikuri informaciis Senaxva da saWiroebis SemTxvevaSi misi gamoyeneba warmoadgens.

pirvelad dnm aRweril iqna 1869 wels f. miSeris mier. Tumca misi funqciis Sesaxeb ujredSi, rogorc memkvidreobiTi informaciis matarebeli makromolekulis Sesaxeb, cnobili gaxda mogvianebiT 1944 wels [1].

1953 wels j. votsonisa da f. krikis mier rentgenostruqturul monacemebze dayrdnobiT SemoTavazebul iqna dnm-is orjaWviani spiraluri sivrculi struqtura (sur. 1), romlis mixedviT dnm-is molekula warmoadgens wrfiv polimers da romlis monomers warmoadgens nukletidi (sur. 2). nukleotidi Sedgeba 5'- an 3'- poziciaSi fosforilirebuli Saqrisgan (dezoqsiribozasagan), romlis C1- atomi dakavSirebulia erT-erT azotovan fuZesTan glikoziduri bmiT C1–N. dnm-Si heterociklur rgolebs warmoadgenen purinebi: adenini (A), Gguanini (G) da pirimidinebi: citozini (C), Timini (T). 3'-5' fosfodieTeruli kavSirebiT nukleotidebis gadabmiT warmoiqmneba dnm-is nukleotiduri jaWvi (sur. 2). rogorc wesi nukleotidebis Tanamimdevrobas jaWvSi kiTxuloben 5'-dan 3'-mimarTulebiT. genetikurad gansazRvruli nukleotidebis Tanamimdevroba gvaZlevs informacias dnm-is molekulis pirveladi struqturis Sesaxeb.

fiziologiur pirobebSi dnm imyofeba ormagspiralur mgdomareobaSi. kavSiri jaWvebs Soris xorcieldeba wyalbaduri kavSirebis saSualebiT azotovan fuZeebs Soris komplementarobis principze dayrdnobiT: yoveli Timini kavSirs warmoqmnis adeninTan ori wyalbaduri kavSiris saSualebiT, xolo yoveli guanini _ citozinTan sami wyalbaduri kavSiris saSualebiT (sur. 2).

pirveli monacemebi dnm-is sivrculi struqturis Sesaxeb miRebuli iyo dnm-is boWkoebis da Txeli firebis rentgenografiuli kvlevebiT marilis sxvadasxva koncentraciis pirobebSi [2-5]. am kvlevebiT naCvenebi iyo, rom dnm warmoadgens marjvena spirals, Tumca mogvianebiT aRmoCenil iqna dnm-is marcxena spiraluri konformaciebic [6]. dnm-is molekulaSi jaWvebis Saqar-fofaturi nawili molekulis zedapirzea gamotanili, warmoqmnian ra koaqsialur marjvena spirals, xolo fuZe wyvilebi ganlagebulia molekulis SigniT, erTmaneTis Tavze.

spiraluri struqturis mqone polinukleotidis daxasiaTeba SesaZlebelia spiralobis parametrebiT. spiralis biji warmoadgens spiralis RerZis gaswvriv manZils, romelic Seesabameba erT srul xvias (sur. 3). Tu nukleotidebis ricxvi erT xviaSi aris n, xolo mezobel nukleotidebs Soris manZili spiralis ReZis gaswvriv _ h, maSin spiralis biji iqneba P=n*h.

rogorc wesi, fuZe wyvilebis centri ar emTxveva spiralis ReZs da wanacvlebulia misgan garkveuli manZiliT (sur. 4). amasTanave wyvilebis sibrtyeebi RerZis mimarT zustad perpendikularuli ar aris. spiralis

6

sur. 1 dnm-is ormagi spiralis sivrculi struqtura

7

zedapiri, cilindris msgavsad, gluv zedapiriani ki ar aris, aramed gaaCnia sxvadasxva siganis da siRrmis ori Rari: mcire da didi (sur. 1).

dReisaTvis dnm-is sami ZiriTadi ojaxia cnobili: A, B da Z (sur. 5) [7,8]. dnm-is B-konformacia bunebaSi yvelaze metad gavrcelebuli formaa. dabali tenianobis garemo pirobebSi dnm-s molekula B-formidan A-formaSi gadadis. ZiriTadi gansxvaveba A- da B-formebs Soris aris dezoqsiribozas cikluri rgolis gansxvavebuli konformacia, romelic B-formaSi imyofeba C2' endo konformaciaSi, xolo A-formaSi C3' endo mdgomareobaSi (sur. 6). amis gamo A-formaSi dnm-is jaWvis gaswvriv nukleotidebs Soris manZili mcirdeba 1 angstremamde. rac Seexeba fuZeebs, A-formaSi isini centraluri RerZidan ufro metad didi Rarisaken arian wanacvlebuli.

Z-forma, B- da A-formebisgan gansxvavebiT, marcxena spirals warmoqmnis. msgavsi struqturebi damaxasiaTebelia purin-pirimidinebis monacvleobiTi Tanamimdevrobis dros, mag. rogoricaa GCGCGC [9-10]. polinukleotidis moTavsebiT MgCl2, NaCl marilebis maRali koncentraciis wyalxsnarSi an spirtSi, SesaZlebeli xdeba marcxena ormagi spiralis miReba. dnm-is am konformaciaSi guanozinis dezoqsiriboza imyofeba C3'-endo konformaciaSi (sur. 6), xolo TviTon guaninis fuZe imyofeba sin-konformaciaSi, gansxvavebiT B da A formebisagan, sadac guanini anti-konformaciaSia (sur. 7). dnm-is sxvadasxva formebis maxasiaTebeli parametrebi moyvanilia cxrilSi 1.

cxrili 1 [11]

ojaxis tipi

forma

spiralis tipi

spiralis mobrunebis kuTxe /grad

fuZe wyvilebis ricxvi spiralis erT xviaSi

TiTo xviis

simaRle

/Å

fuZeebis daxris kuTxe

spiralis RerZisadmi

/grad

ganmeorebadi

erTeulebis

proeqcia spiralis RerZze

/Å

ormagi spirali

s diametri

/Å

A

A12

A11

A

kanonikuri

marjvena

spirali

igive

igive

30.0

32.7

33.6

12

11

10.7

28.2

-10

-19

-19

3.0

2.6

2.3

23

B

B

C

D

igive

igive

igive

35.9

38.6

45.6

10.5

9.3

8

33.2

6

8

16

3.38

3.32

3.04

20

Z

kanonikuri

marcxena

spirali

60/2 12/6 45.6 3.8 18

8

sur. 2 dnm-is ormagi nukleotiduri jaWvis sqematuri suraTi

9

sur. 3 dnm-is mezobel nukleotidebs Soris manZili da spiralis bijis simaRle

sur. 4 fuZe wyvilebis centrebi wanacvlebuli spiralis RerZidan

10

sur. 5 dnm-is A, B, da Z-formebi

11

sur. 6 dezoqsiribozas ciklur rgolSi C2'-endo konformaciaSi da C3'-endo mdgomareobaSi

sur.7 guaninis anti- da sin-konformaciebi

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funqciuri TvalsazrisiT ujredis dnm-is grZel polipeptidur jaWvSi gamoyofen sxvadasxva daniSnulebis mqone ubnebs. dnm-is im monakveTs, romelic ganapirobebs cilebis, t-rnm-is da r-rnm-is sinTezs geni ewodeba. ganasxvaveben ori tipis genebs: struqturuls da maregulirebels. struqturuli genebi gansazRvraven genis saboloo produqtis, cilis an rnm-is, pirvelad struqturas. zogierTi maTgani SedarebiT ufro xSirad transkribirdeba (housekeeping genes). amasTanave dnm-is molekulaSi gvxvdeba SedarebiT iSviaTad transkribirebadi genebic, romelTa eqspresia sxvadasxva faqtorebzea damokidebuli (garemos zemoqmedeba, ganviTarebis stadia, ujredis specializacia da a.S.). yvela aseT makodirebel ubnebs egzonebs uwodeben. rac Seexeba maregulirebel genebs, isini xSirad warmoadgenen struqturuli genis daboloebis an dasawyisis signals, romlebic monawileoben am genebis transkrifciis procesSi, aseve gansazRvraven replikaciis an rekombinaciis sawyis wertilebs. yoveli genis dasawyisSi mdebareobs transkrifciis mainicirebeli nukleotidebis specifiuri Tanamimdevroba, romelsac promotors uwodeben. rogorc cnobilia E.Coli-is promotorebi e. w. konsensuri TanamimdevrobebiT xasiaTdebian. aseT TanamimdevrobebSi gamoyofen specifiur: –10 ubans –TATAAT (TATA-box) da –35 ubans – TTGACA. Tumca arakonsensuri genebis SemTxvevaSi es Tanamimdevroba metnaklebad SeiZleba gansxvavdebodes.

E. Coli –is promotorebis konsensuri Tanamimdevrobebi

is promotorebi, romelTa Tanamimdevroba zustad emTxveva konsensur Tanamimdevrobebs, transkrifciis iniciaciis maRali sixSiriT xasiaTdebian. amitomac mutaciebi, romlebsac adgili aqvT promotorul ubnebze, iwveven am ubnis promotoruli funqciis gaZlierebas an Sesustebas. cnobilia, rom genebi, romelTa promotorebSi A an T

nukleotidis Cveul poziciaSi G an C gvxvdeba sust genebs miekuTvneba da isini naklebi sixSiriT transkribirdebian [12]. amave dros transkribciis sixSire damokidebulia _35 da _10 fragmentebs Soris manZilze. Tu gaviTvaliswinebT dnm-is ormagspiralur struqturas, am fragmentebs Soris manZilis cvilebis (gazrda an Semcireba) Sedegad SesaZlebelia isini dnm-is jaWvis erT mxares aRar aRmoCndnen da rnm polimerazas orive maTganTan erTdroulad kontaqtis unari mcirdeba.

garda _35 da _10 ubnebisa promotoruli Tanamimdevrobis sxva ubnebic axdenen gavlenas transkribciis sixSireze. yvelaze aqtiurni arian promotorebi, romlebic warmarTaven ribosomuli rnm-is operonebis transkribcias. _35 da _10 ubnebis garda isini Seicaven aseve mesame elements, AT-wyvilebiT mdidar Tanamimdevrobebs, e.w. UP elementebi, romlebic 30-90-jer zrdian transkribciis sixSires. garda amisa, promotorul aqtivobaze gavlenas axdens dnm-is lokaluri struqturis cvlileba. mag. dnm-is zespiralizacia da moxra (moRunva) gavlenas axdens rnm polimerazas mier ormagi spiralis gaxsnis unarze.

eukariotuli ujredebis SemTxvevaSi, prokariotebisTvis damaxasiaTebeli AT-wyvilebiT mdidari ubnis analogiuria e.w. TATA-box

13

ubani, romelic mdebareobs rnm-is sinTezis iniciaciis adgilidan wakiTxvis sawinaaRmdego mimarTulebiT, 19-dan 27-mde fuZe wyvilebs Soris intervalSi.

ganzogadebuli eukariotuli promotorebi.

genis 3'- boloSi arsebul specifiur ubanze (terminaciuli ubani) dnm-cilis kompleqsi arastabiluri xdeba, risTvisac zogierT SemTxvevaSi aucilebeli xdeba damatebiT specifiuri cilebis arsebobac. transkrifciis terminacia xSirad xdeba Semayovnebeli saitis (Pause site) axlos, sadac adgili aqvs transkrifciis siCqaris Semcirebas (10–100 faqtoriT). literaturuli monacemebidan cnobilia, rom aseTi ubnebi mdidaria GC - wyvilebiT, rac savaraudod ganpirobebulia GC wyvilebs Soris kavSirebis siZlieriT AT- wyvilebTan SedarebiT [12].

transkrifciis mainicirebeli ubnis – promotoris msgavsad, rogorc prokariotebis, ise eukariotebis dnm-Si gamoyofen replikaciis iniciaciis adgils. igi maregulirebeli tipis genebs ganekuTvneba. E.Coli baqteriis SemTxvevaSi aseT ubans warmoadgens e. w. oriC (Origin of replication E.Coli), romelic 245 fuZe wyvilisagan Sedgeba. baqteriuli ujredis dnm-is am monakveTSi funqciurad gansakuTrebiT mniSvnelovania ori tipis nukleotiduri Tanamimdevroba: pirveli maTgani Sedgeba 3-jer ganmeorebadi 13 fuZe wyvilisagan, xolo meore – 4-jer ganmeorebadi 9 fuZe wyvilisagan [13,14]. rogorc literaturuli monacemebidan aris cnobili, dnm-is jaWvebis gancalkevebis sawyis ubans warmoadgens 13 fuZe wyvilisagan Semdgari Tanamimdevroba, romelic AT- wyvilebiTaa mdidari [15,16]. Tumca zusti bioqimiuri meqanizmi, romliTac jaWvebis gancalkeveba miiRweva jer-jerobiT ucnobia.

garda amisa, eukariotuli ujredebis umravlesobis genSi arsebobs iseTi segmentebi, romlebic saerTod ar akodireben cilebs an rnm da isini genis wyvetilobiT xasiaTs ganapirobeben. aseT aratranskribirebad segmentebs intronebi ewodeba. eukariotuli dnm-is polipeptiduri jaWvisTvis damaxasiaTebelia aseve sxvadasxva sixSiriT ganmeorebadi fragmentebis arseboba. am segmentebs satelitur dnm-sac uwodeben. maTi funqcia bolomde Seswavlili ar aris da Tanamedrove mecnierTaTvis aqtiuri kvlevis sagans warmoadgens. cnobilia, rom adamianis dnm-is daaxloebiT 95% cilebs an rnm-s ar akodirebs, Tumca maT gaaCniaT regulatoruli da sxva funqciebi, romelTa Seswavla aseve intensiurad mimdinareobs.

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1.2. dnm-is ormag-spiraluri struqturis mastabilizirebeli

Zalebi

dnm-is struqturis mastabilizirebel Zalebs da faqtorebs

miekuTvneba: wyalbaduri kavSirebi wyvilebs Soris, steking urTierTqmedeba nukleotidur jaWvSi mezobeli fuZeebis sibrtyeebs Soris [17], dnm-is fosfaturi jgufebis eleqtrostatikuri urTierTqmedeba garemomcvel ionebTan [18], fuZeebis hidrofobuli buneba, bmuli wylis gavlena.

ganvixiloT TiToeuli maTgani cal-calke. sazogadod, rogorc cnobilia wyalbaduri kavSiri xorcieldeba

Zlier eleqtrouaryofiT atomTan kovalenturad dakavSirebul wyalbadis atomsa da daaxloebiT 0.2 nm manZilze daSorebul sxva eleqtrouaryofiT atoms Soris, romelsac gaaCnia gauwyvilebeli eleqtroni. azotovani fuZe wyvilebis SemTxvevaSi, wyalbadis atomi kavSirs axorcielebs eleqtro-uaryofiT azotsa da Jangbads, an azotis atomebs Soris. wyalbaduri bmis energia, romelic dnm-Si azotovan fuZe wyvilebs Soris xorcieldeba daaxlovebiT 2-7.5 kcal/mol-ia [19].

dnm-is molekulaSi azotovani fuZeebis aromatuli rgolebi erTmaneTis Tavze dnm-is centraluri RerZis mimarT TiTqmis perpendikularuladaa ganlagebuli. maTi aseTi urTierTganlageba ki

uzrunvelyofs steking urTierTqmedebas mezobel fuZeebs Soris, romelic damatebiTi mastabilizirebeli Zalaa dnm-is ormagi spiralisaTvis. am tipis urTierTqmedeba miekuTvneba arakovalentur qimiur bmas. miuxedavad imisa, rom arakovalenturi bmebi gacilebiT sustia, vidre kovalenturi tipis kavSirebi, dnm-is jaWvSi ganxorcielebuli steking urTierTqmedebebis erToblioba saboloo jamSi ormagi spiralis mniSvnelovan mastabilizirebel Zalas warmoadgens. steking urTierTqmedebis energia sxvadasxva azotovan fuZeebs Soris gansxvavebulia (ix. cxrili 2). am monacemebidan kargad Cans, rom energetikulad yvelaze sust kavSirs (ΔG= - 0.19 kcal mol

-1) TA

nukleotiduri Tanamimdevroba warmoqmnis, rac metad sayuradReboa.

cxrili 2

biji

steking urTierTqmedebis

energia ΔG

/kcal mol-1

T A -0.19

T G an C A -0.55

C G -0.91

A G an C T -1.06

A A an T T -1.11

A T -1.34

G A an T C -1.43

C C an G G -1.44

A C an G T -1.81

G C -2.17

15

aseve Zlier gavlenas axdens dnm-is ormagi spiralis stabilobaze

marilebis koncentraciis cvlileba. dnm-is uaryofiTad damuxtuli jgufebis (fosfaturi naSTebi) ekranireba sapirispiro muxtis mqone ionebiT ganapirobeben jaWvebs Soris eleqtrostatikuri ganzidvis Semcirebas da dnm-is ormag-spiraluri struqturis warmoqmnas [21]. eleqtrostatikuri ekranirebis garda marilis maRali koncentracia mniSvnelovan rols TamaSobs dnm-is mimarT wylis aqtivobis SemcirebaSi. marilis maRali koncentraciisas spiralis daxvevis kuTxe Seqcevadad icvleba [20].

mravali eqsperimentuli monacemebiT naCvenebia wylis gansakuTrebuli roli nukleinis mJavebis konformaciis formirebaSi (sur. 8) [22-28]. mniSvnelovania, aseve, calkeuli wylis molekulis struqturuli roli dnm-ze, romelic sxvadasxva nukleotiduri TanamimdevrobisaTvis sxvadasxvagvarad vlindeba. dnm-is kalorimetruli kvlevebis mixedviT aRmoCnda, rom AT- wyvilebi metadaa hidratirebuli, vidre _ GC wyvilebi [29,30].

gamxsnelSi dnm-is molekula aramdgradia da misi struqtura mudmivad ganicdis garkveul fluqtuaciur cvlilebebs. cnobilia, rom aseT pirobebSi dnm-Si adgili aqvs ormagi jaWvis droebiT lokalur gancalkevebebs [31]. dnm-is molekula aseve ganicdis brunviT rxevebsac, kerZod, superspiraluri dnm-is kvlevebis Teoriulma analizma aCvena, rom jaWvis gaswvriv or mezobel wyvilebs Soris kuTxis amplitudis fluqtuacia 50-s aRwevs [32].

dnm-is mowesrigebuli struqturis stabiloba SeiZleba gansazRvruli iyos Tavisufali energiis cvlilebiT spiral-gorgaluri gadasvlisas. kalorimetrulad gazomili enTalpiis da entropiis cvlileba dnm-is aseTi konformaciuli gadasvlebisas [33], saSualebas iZleva Tavisufali energiis cvlilebis gansazRvrisa, amis gamo naSromSi kalorimetria erT-erT ZiriTad meTodad aris warmodgenili.

16

sur. 8 dnm-is mcire RarSi arsebuli bmuli wyali

17

1.3. dnm-is spiral-gorgaluri gadasvla

cnobilia, rom sxvadasxva gareSe faqtorebis (temperatura,

erTvalentiani da orvalentiani ionebis arseboba, garemos pH, sxvadasxva metalebis gavlena da a.S.) zemoqmedebisas dnm-is ormagspiraluri struqtura ganicdis konformaciul cvlilebebs [34-39]. madestabilizirebeli zemoqmedebis gaZlierebasTan erTad dnm-is jaWvebi iwyeben jer nawilobriv, xolo Semdeg mTlianad gancalkevebas (denaturacia). am process, xSirad, spiral-gorgalur fazur gadasvlasac uwodeben (sur. 9) [40]. gamomdinare iqidan, rom AT da GC wyvilebi sxvadasxva TermostabilobiT xasiaTdebian (fiziologiur pirobebSi), kerZod AT- wyvilebi naklebad stabiluria (ori wyalbaduri kavSiri), vidre - GC (sami wyalbaduri kavSiri), dnm-is lRoba AT- wyvilebiT mdidari ubnebidan daiwyeba. Sedegad dnm-is denaturaciuli gadasvlis procesSi nawili molekulisa imyofeba spiralur da nawili denaturirebul mdgomareobaSi [41]. Tuki dnm-is ormagi spirali warmoadgens sakmaod xist struqturas, persistentuli sigrZiT _ 150 fuZe wyvili, galRobili (gancalkevebuli jaWvebi) ubnebi dnm-sa, piriqiT, sakmaod maRali drekadobiT gamoirCevian (persistentuli sigrZiT _ 4 fuZe wyvili). garemos parametrebis mcire Secvla, romlis drosac xdeba wyalbaduri kavSirebis garkveul adgilebSi darRveva fuZeebs Soris da amis Semdeg Tu molekulas isev sawyis pirobebSi davabrunebT, moxdeba molekulis komplementarul wyvilebs Soris darRveuli wyalbaduri kavSirebis xelaxla aRdgena, anu dnm-is molekula aRidgens pirvandel ormag-spiralur struqturas. zogadad, dnm-is spiral-gorgaluri gadasvla garkveul garemo pirobebSi ("susti" destabilizacia) Seqcevadi xasiaTisaa.

vinaidan dnm-is spiral-gorgaluri (denaturacia) gadasvlis process Tan sdevs dnm-is maxasiaTebeli mravali fizikuri parametrebis cvlileba, misi Seswavla sxvadasxva meTodebiTaa SesaZlebeli. cnobilia, rom Tu temperaturis zemoqmedebiT vaxdenT spiral-gorgalur (denaturacia) gadasvlas, am process Tan axlavs siTbos STanTqma, romelic xasiaTdeba gadasvlis enTalpiis da entropiis garkveuli mniSvnelobebiT. dnm-is

galRobili nawilis (1-) temperaturaze damokidebulebis mrudi naCvenebia nax. 1-ze. mis erT-erT maxasiaTebels warmoadgens denaturaciuli

gadasvlis temperaturuli intervali (Tm), romlis mixedviTac SeiZleba vimsjeloT am procesis kooperatulobaze. dnm-is lRobis tipiuri mikrokalorimetruli Canaweri moyvanilia nax. 2-ze, sadac sakvlevi nimuSis siTbotevadobis temperaturaze damokidebulebaa mocemuli. am SemTxvevaSi dnm-is lRobis temperaturul intervals gansazRvraven, rogorc siTbos STanTqmis pikis siganes naxevarsimaRleze, xolo gadasvlis enTalpia ganisazRvreba pikis qveS miRebuli farTiT. mravali eqsperimentuli SedegebiT naCvenebia, rom dnm-is lRobis temperatura damokidebulia garemo pirobebze [42,43]. aseve naCvenebia [44,45], rom fazuri gadasvlis (lRobis) temperatura damokidebulia dnm-Si GC wyvilebis Semcvelobaze da warmoadgens wrfiv funqcias, romelic ganisazRvreba formuliT:

%GC=(Tm-69,3)·2,44

18

sur. 9 dnm-is spiral-gorgaluri gadasvla

19

eqsperimentulad gansazRvrulia, rom dnm-is lRobis temperatura mniSvnelovnadaa damokidebuli garemo pirobebze [46], kerZod neitraluri pH-is gamxsnelis da Na-is koncentraciis 10-2M-dan 1M-mde intervalis pirobebSi es damokidebuleba ganisazRvreba rogorc

T0=176.0-(2.6-X0)·(36.0-7.04 lg[Na

+])

sadac X0 _ GC wyvilebis wilia.

20

nax. 1 dnm-is lRobis mrudi

nax. 2 ormagspiraluri dnm-is lRobis kalorimetruli mrudi

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

8

Cp

[Jg

-1K

-1]

T [ 0C]

21

1.4. dnm-is ormagi spiralis protonizacia/deprotonizacia

literaturuli monacemebidan cnobilia, rom pH~7.0 -is pirobebSi

dnm-is fuZe wyvilebi ionizebuli ar arian [47]. amave pirobebSi protonizacias aseve ar ganicdis fosfaturi jgufebi, romelTaTvisac pK~1, da dezoqsiriboza, romlis pK~12-13.

pH-is SemcirebiT, anu protonizaciis gazrdiT, protonis mierTeba xdeba azotovani fuZeebis im atomebTan, sadac meti uaryofiTi muxtia

koncentrirebuli da rasac -eleqtronebi ganapirobeben. am atomebs gaaCniaT gauwyvilebel eleqtronebi, rac Tavis mxriv aadvilebs protonis mierTebas. am mosazrebebze dayrdnobiT protonizaciis SesaZlo adgilebs warmoadgens citozinis N3, guaninis N7 da adeninis N1, Tumca rogorc eqsperimentuli monacemebiT dasturdeba dnm-is ormagspiralur struqturaSi protonizacias ganicdis mxolod N7 guanini da N1 adenini [48-57]. es SeiZleba ganpirobebuli iyos im faqtiT, rom dnm-is ormag spiralSi, votson-krikis fuZe wyvilSi, guaninis N7 mdebareobs ormagi spiralis did RarSi da igi xelmisawvdomia protonisTvis, maSin roca citozinis N3 CarTulia GC fuZeebs Soris ganxorcielebul erT-erT wyalbadur bmaSi [58], ris gamoc misi protonireba ar xdeba. literaturuli monacemebis mixedviT [57] citozinis protonizacias adgili aqvs GC wyvilis proton-inducirebuli konformaciuli cvlilebebis ganxorcielebis Semdeg, rac gulisxmobs guaninis N7-dan citozinis N3-ze protonis gadatanas. amas adasturebs is faqtic, rom citozinis protonizacia SeuZlebelia ganxorcieldes, roca guaninis N7 centri blokirebulia romelime ligandiT. amrigad, GC fuZeebis sami SesaZlo protonizaciis centridan dnm-is ormag spiralSi realuri protonizaciis adgilebad miCneulia N7 guanini da N1 adenini.

literaturaSi arsebuli monacemebis Tanaxmad pH-is SemcirebiT SesaZlebelia moxdes dnm-Si GC wyvilebiT mdidari adgilebis axal konformaciaSi gadasvla [59-62], romelic avtorebis mier interpretirebuli iqna rogorc B-dan C-konformaciaSi gadasvla. guaninis N7-is protonireba iwvevs hetero-ciklur rgolze uaryofiTi muxtis Semcirebas, rac Tavis mxriv asustebs wyalbadur kavSirebs azotovan fuZeebs Soris(G da C). rogorc literaturuli monacemebidan aris

cnobili [63-65], am dros xdeba GC wyvils Soris arsebuli N1-H···N3 da N2-

H···O wyalbaduri bmebis gawyveta, glikoziduri bmis garSemo mobrunebis xarjze guaninis azotovani fuZis anti-dan sin-konformaciaSi gadasvla, ris Sedegadac warmoiqmneba e. w. hugstinis GC wyvilebi ori wyalbaduri kavSiriT (ix. sur. 10). protonizaciis Sedegad miRebuli axali tipis GC wyvili naklebad stabiluria, magram miuxedavad amisa dnm-is meoradi struqtura jer kidev SenarCunebuli rCeba. avtorebis [58] mier naCvenebi iqna, rom AT- wyvilebis protonizacia, xdeba uSualod dnm-is denaturaciis dawyebis win, romlis drosac AT- wyvilebs (adeninis N6-sa da Timinis C4-s) Soris mxolod erTi wyalbaduri kavSiria SenarCunebuli. es wyvili ufro arastabiluri unda iyos, vidre protonirebuli GC

wyvili, sadac ori wyalbaduri kavSiri rCeba. logikuria vivaraudoT, rom mJavaTi denaturaciisas dnm-Si yvelaze naklebad stabiluri AT- wyvilebiT mdidari ubnebia.

fluorescenciuli kvlevebis mixedviT [58], AT-fuZe wyvilebis protonizacia iwyeba pH~6.0-dan, xolo mJavaTi inducirebuli dnm-is konformaciuli cvlilebebi pH<5.0. ramanis speqtroskopiT miRebuli

22

sur. 10 votson-krikisa da e.w. hugstinis fuZe wyvilebi

23

monacemebis mixedviT [64], kvlevebis Tanaxmad, GC- wyvilebis

protoniazacia ufro dabal pH-ze (~3.8) adeninis protonizacia SesamCnevi xdeba pH 4.4 da grZeldeba pH 3.45-mde, rac dakavSirebulia AT- wyvilebs Soris kavSirebis rRvevasTan. amave iwyeba da grZeldeba pH 3.45-mde.

wriuli diqroizmis da kinetikuri meTodiT kvlevebis mixedviT naCvenebia, rom mJava garemoSi warmoiqmneba dnm-is stabiluri protonirebuli e.w. "S" struqtura, sadac azotovani fuZeebi mTlianad mowesrigebulia [67-69]. am dros adgili aqvs guaninis Seqcevad gadasvlas anti-dan sin-konformaciaSi, anu arastabilur e.w. "L" mdgomareobaSi, romlis drosac rogorc varaudoben dnm-s gaaCnia ormagspiraluri struqtura lokalurad denaturirebuli (gancalkevebuli) ubnebiT [68,69]. Tumca am struqturis buneba jer kidev bolomde garkveuli ar aris.

eqsperimentulad naCvenebia, rom mJave areSi adgili aqvs AT da GC

wyvilebis Termostabilobis Semcirebas da erTmaneTTan daaxlovebas [71], Tumca naCvenebia, rom dnm-is xsnarSi tute metalebis koncentraciis gazrdiT dabali pH-is pirobebSi, iseve rogorc neitraluri pH-is pirobebSi, makromolekulis stabiloba izrdeba. Teoriulad da eqsperimentulad (ZiriTadad speqtrofotometruli meTodiT) naCvenebi iqna [72,73], rom tute areSi, rac ufro didia gamxsnelis ionuri Zala, miT naklebia dnm-is lRobis temperatura. unda aRiniSnos, rom neitraluri pH-is pirobebis msgavsad, dnm-is ormagi spiralis Termuli stabiloba mJave garemo pirobebSi ionuri Zalis gazrdisas aseve matulobs. amas xsnian im faqtiT, rom sxnarSi metalis ionebis arseboba mkveTrad amcirebs im jgufebis (guaninis N1 da Timinis N3) pK-s mniSvnelobas, romlebic tute areSi deprotonizacias ganicdian. rac Seexeba wyvilebis protonizaciis unars, naCvenebia rom ionuri Zala sust gavlenas axdens am procesze [48].

mJave aresagan gansxvavebiT SedarebiT mwiria informacia dnm-is Tvisebebis Sesaxeb tute areSi, gansakuTrebiT ki sust tute garemoSi (pH

8.0-10.0). literaturuli monacemebis Tanaxmad [48], dnm-is molekulaSi azotovani fuZeebis deprotonizacias (pH>7) adgili aqvs guaninis N1-ze da Timinis N3-ze.

speqtrofotometruli gazomvebiT naCvenebia, rom dnm-is titraciisas tute arisken, sanam dnm-s jer kidev SenarCunebuli aqvs ormag-spiraluri struqtura, fuZeebis deprotonizacia ar xdeba an Zalian mcirea [74-76]. amave kvlevebis Tanaxmad, tuteTi inducirebuli dnm-is meoreuli struqturis konformaciuli cvlilebebi ZiriTadad Tavs iCens pH>11.6 dros [74]. am cvlilebebisas ukve iwyeba fuZeebis ionizacia da maT Soris wyalbaduri kavSirebis Semcireba, rasac Tan sdevs dnm-is meoreuli struqturis rRveva [77]. avtorebis [77] mixedviT, dnm-is tute denaturacia unda xorcieldebodes hidroqsilis ionebis mier fuZe wyvilebs Soris wyalbadur kavSirSi monawile wyalbadis atomis mowyvetiT da masTan kavSiriT wylis molekulis warmoqmniT. dnm-is viskozimetruli SeswavliT tute areSi (pH>8.0) naCvenebia [79], rom maRali ionuri Zalis pirobebSic dnm-is sixiste izrdeba, rac miuTiTebs aseT garemoSi dnm-is struqturul stabilobaze. aseve cnobilia, rom ~pH 12.5 tutianobis pirobebSi dnm-is molekula mTlianad denaturirebul mdgomareobaSi imyofeba.

24

1.5. specifiuri da araspecifiuri urTierTqmedebis saxeebi

(tipebi) dnm-is jaWvTan

genetikuri informaciis xelmisawvdomoba damokidebulia cilebis

unarze amoicnon dnm-is garkveuli ubani da moxdes maT Soris urTierTqmedeba iseTnairad, rom SesaZlebeli gaxdes dnm-is nukleotidur jaWvSi kodirebuli informaciis kopireba dnm-iT (replikacia), an rnm-iT (transkrifcia). umartivesi “nabijebic” ki am procesisa moiTxovs mraval specifiur cilas, romlebic urTierTqmedeben erTmaneTTan da nukleinis mJavebTan (specifiur ubnebTan). amasTanave organizms SeuZlia areguliros genebis umetesobis eqspresia, romelic kidev sxva tipis cilebis arsebobasac saWiroebs da moqmedeben rogorc transkrifciis aqtivatorebi an represorebi.

mravali cila, magaliTad histonebi, araspecifiurad ukavSirdeba dnm-s, anu ganurCevlad imisa Tu rogoria nukleotidebis Tanamimdevroba. dadgenilia aseve, rom dnm-is replikaciaSi monawile zogierTi tipis cila, funqcionalur jgufebis meSveobiT eleqtrostatikuri urTierTqmedebis saSualebiT kavSirs warmoqmnian dnm-is fosfatur jgufebTan (araspecifiuri urTierTqmedeba). cilebi, romlebmac unda amoicnos dnm-is specifiuri nukleotiduri Tanamimdevroba, Tavdapirvelad, savaraudod aseve araspecifiurad da sustad ukavSirdebian dnm-s, raTa polinukleotiduri jaWvis gaswvriv “moZraobiT” SeZlon Sesabamisi specifiuri ubnis povna. ZiriTadi mainicirebeli Zala sxvadasxva makromolekulebis dnm-Tan araspecifiuri urTierTqmedebisa eleqtrostatikuri xasiaTisaa da emyareba dnm-is uaryofiTad damuxtul fosfatur jgufebsa da masTan urTierTqmed makromolekulis dadebiTad damuxtul jgufebs Soris kavSiris warmoqmnas. urTierTqmedebis msgavsi meqanizmi udevs safuZvlad genis gadamtani sxvadasxva biopolimeruli nawilakisa da Sesabamisi genuri masalis kompleqsis warmoqmnas. es sistemebi (nano zomis nawilakisa da dnm (genis) kompleqsi) ukve gamoiyeneba genur TerapiaSi, Tumca jer kidev aqtiurad mimdinareobs kvlevebi am mimarTulebiT [80,81,82]. genoTerapia gulisxmobs cocxali organizmis ujredSi oligo- an polinukleotiduri masalis Seyvanas arasasurveli genebis funqciebis dasaTrgunad, an Sesabamisi genebis eqspresiis mizniT, imisaTvis, rom moxdes gamomuSaveba ar arsebuli, an arasakmarisi raodenobiT myofi produqtebisa. amitom saWiro oligonukleotidma unda miaRwios gansazRvrul qsovilebs, an organoebs, gadakveTos plazmaturi membrana imisaTvis, rom ujredis SigniT gansazRvrul samizne ubanSi (birTvSi) moxvdnen da ipovnon is m-rnm-is, an qromosomuli dnm-is ubani, romlis winaaRmdegac isini arian mimarTulni. Seyvanili geni unda Sevides misi produqtis eqspresiis unaris mqone konstruqciis SemadgenlobaSi. radganac gens, an oligonukleotids TavisTavad es Tvisebebi ar gaaCniaT, organizmSi Seyvanisas mxolod maTi mcire raodenoba miaRwevs saWiro adgils. ujredSi moxvedrisas ucxo dnm-ma SeiZleba ar moaxdinos lokalizacia saWiroebis adgilze da ufro metic, SeiZleba aRmoCndes lizosomebSi, sadac nukleazebis zemoqmedebiT SeiZleba moxdes maTi daSla. dnm-is molekulis CarTva sxvadasxva tipis nano zomis polimerul nawilakSi icavs mas organizmSi arsebuli nukleazebisagan [83].

dReisaTvis damkvidrebuli mosazrebiT cila-dnm-s Soris specifiuri urTierTqmedeba ZiriTadad konformaciul Sesabamisobas emyareba. sxva avtorebis Tanaxmad [84-87] dnm-Tan specifiurad dakavSirebuli cilebi, sazogadod, ar arRveven dnm-is ormagi spiralis

25

bazisur wyvilebs Soris kavSirs da oTxi bazisuri wyvilis (AT, TA, GC da CG) kombinaciiT Sedgenili specifiuri ubnis amocnoba xdeba am ukanasknelTa funqionaluri jgufebis mixedviT, romlebic gamotanili arian dnm-is did an mcire RarSi. amis Semdeg cila urTierTqmedebs did da mcire RarSi eqsponirebuli fuZe wyvilebis funqcionalur jgufebTan, ris Sedegadac warmoiqmneba cila-dnm-is Caketili kompleqsi (dnm-is jaWvebs Soris kavSiri araa darRveuli) [88,89]. miRebuli cila-dnm-is kompleqsSi ganxorcielebuli konformaciuli cvlilebebis Sedegad dnm-is jaWvebi gancalkevdeba da miiReba Ria kompleqsi [88]. cilis mier dnm-is ubnis amocnobis aseT scenars iziarebs mravali mecnieri, Tumca, rogorc zemoT avRniSneT, procesis aseTi TanmimdevrobiT ganxorcielebis Sesaxeb utyuari damadasturebeli monacemebi dRes-dReobiT ar arsebobs. amave dros gaugebaria Tu raSi mdgomareobs dnm-is amaTu im bazisuri wyvilis unikaluroba, funqcionaluri jgufebis TvalsazrisiT, vinaidan dnm-is amomcnobi cila igive warmatebiT SeiZleba daukavSirdes sxva mcire an did RarSi eqsponirebul igive tipis azotovani fuZis funqcionalur jgufs dnm-is jaWvis gaswvriv.

jer-jerobiT zustad dadgenili ar aris im urTierTqmedebis Zalebis buneba, romlebic safuZvlad udevs cilis mier dnm-is ubanTan specifiuri kompleqsis warmoqmnas. gazomili iqna aseTi urTierTqmedebis (dnm-cilis kompleqsi) disociaciis konstantis mniSvneloba, romelic aRmoCnda 10-9-10-12 M

-1 rigis, romelic dnm-cilis araspecifiuri urTierTqmedebis konstantis mniSvnelobaze 103-107 jer metia.

literaturidan cnobilia, rom arahistonuri cilebi (mJave cilebi) Seicaven iseT domenebs, romliTac specifiurad ukavSirdebian dnm-is garkveul ubnebs, riTic xels uwyoben transkrifciis procesis aqtivacias, anu aseTi cilebi monawileoben rnm-is sinTezis pozitiur kontrolSi [90-95].

cnobilia, rom dnm-Tan specifiuri urTierTqmedebiT gamoirCeva rnm polimeraza, romelic warmoadgens erT-erT umniSvnelovanes biomakromolekulas da romelic mTavar rols TamaSobs rogorc eukariotuli, ise prokariotuli ujredis genuri transkrifciis procesSi. naCvenebia, rom eukariotul ujredSi rnm polimeraza promotorebTan (AT-wyvilebiT mdidari ubnebi dnm-is polinukleotidur jaWvSi, e.w. TATA-box) erTad warmoqmnis stabilur kompleqss, romelic uzrunvelyofs transkrifciis zust da efeqtur dawyebis adgilis gansazRvas (sur. 11) [96-101]. rnm- polimeraza ukavSirdeba nukleozid trifosfatis substrats da akatalizebs rnm-is sinTezs, xolo specifiur adgilas axdens rnm-is sinTezis terminacias, rogorc varaudoben terminaciul faqtorebTan urTierTqmedebiT. dadgenilia, rom rnm polimerazas modifikacia pasuxismgebelia genuri eqspresiis regulaciazec [102-104]. vinaidan rnm-polimerazas funqciuri SesaZleblobebi ganpirobebulia misi Semadgeneli suberTeulebis struqturiT, am struqturebis detaluri kvleva da calkeuli suberTeulis struqturis codna saSualebas mogvcems transkrifciis procesis da misi regulaciis meqanizmebis ukeT garkvevaSi.

rogorc cnobilia, prokariotul ujredebSi erTi tipis rnm-polimeraza axorcielebs yvela rnm-is sinTezs. igi Sedgeba ZiriTadi

fermentisa (',suberTeulebi) da specifiuri faqtorisagan, romelTa kombinaciac (holoenzimi) gansazRvravs Tu romeli promotori

unda transkribirdes dnm-is jaWvSi. cnobilia, rom kDada '(156 kDa)

suberTeulebi mniSvnelovnad didia vidre (37 kDa) da (11 kDa).

26

sur. 11 rnm polimeraza-dnm-is urTierTqmedeba da transkrifciis procesis warmarTva

27

baqteriul ujredebSi ramodenime sxvadasxva formis suberTeuli iqna identificirebuli, molekuluri woniT 28 kDa-dan 70 kDa-mde. isini

droebiT ukavSirdebian ZiriTad ferments (',), mniSvnelovan rols TamaSoben transkrifciis iniciaciis adgilis amocnobaSi da aseve gaaCniaT helikaza aqtivoba dnm-is ormagi spiralis gaSlisa. cnobilia,

rom im SemTaxvevaSi Tu ZiriTadi fermenti araa dakavSirebuli -sTan, igi araspecifiurad ukavSirdeba dnm-s, anu misi swrafva promoterisken igivea

rac dnm-is sxva Tanamimdevrobebisken (Kassoc.≈10-10

M-1), xolo cila-dnm-is

kompleqsis disociaciis dro ~ 60wT-ia. roca ZiriTadi fermenti

dakavSirebulia -faqtorTan, misi swrafva dnm-is promoteruli ubnisken

mniSvnelovnad izrdeba (Kassoc.≈2·10-11

M-1) da warmoqmnis ufro stabilur

kompleqss (t1/2≈2-3 h)[12]. transkrifciis iniciaciis procesi, romelic genis eqspresiis sawyis

etaps warmoadgens, gulisxmobs promotoris "lRobas" (jaWvebis gancalkevebas) ise, rom rnm polimerazasTvis xelmisawvdomi gaxdes dnm-is ormagi spiralis is jaWvi (e.w. sayalibo jaWvi), saidanac saWiroa informaciis wakiTxva. aRsaniSnavia, rom promotoris “lRobis” meqanizmi jer-jerobiT ucnobia. am sakiTxTan dakavSirebiT sami urTierT aragamomricxavi modeli iqna SemoTavazebuli: dnm-s ganxvevis (untwisting), dnm-s moxris (bending) da azotovani fuZeebis mobruneba glikoziduri bmis irgvliv (base flipping) (fuZeebis dnm-is spiralSi CaWera an spiralis gareT eqsponireba)[105]. iungis da sxvebis mier naCvenebi iqna, rom Esherichia coli-is rnm polimerazas molekulis mxolod mcire nawils (<1/5), gaaCnia unari

baqteriul iniciaciur faqtor -sTan erTad promotoris galRobisa. transkrifciis regulacia eukariotuli ujredis birTvSi

SedarebiT rTuli multikomponentiani cila-dnm-is kompleqsis warmoqmnas moiTxovs [106]. am SemTxvevaSi cilebis kompleqsi, romelic rnm polimerazasTan erTad moicavs mraval transkrifciul faqtors, mniSvnelovani specifiurobiT cnobs dnm-is mokle ubnebs. sazogadod, cilebis am rTuli kompleqsis warmoqmna ganisazRvreba transkrifciuli faqtorebiT, romelic dnm-is gareSe ar mimdinareobs. ufro metic, zogierTi individualuri faqtoris swrafva dnm-Tan dakavSirebisa sxva cilis komponentebis gareSe SeiZleba sakmaod susti iyos. am kompleqsis warmoqmna savaraudod mimdinareobs kompleqtirebis modeliT, romlis mixedviTac dnm moqmedebs, rogorc yalibi, sadac komponentebi TanamimdevrobiT ukavSirdebian erTmaneTs da warmoqmnian agregatebs.

garkveuli tipis cilebis mier dnm-is specifiuri Tanamimdevrobebis amocnoba xdeba aseve replikaciis dros. rogorc wina TavSi avRniSneT, E.Coli -is SemTvevaSi dnm-is amocnobis da replikaciis dawyebis (jaWvebis gancalkeveba) adgils warmoadgens oriC, sadac moTavsebulia nukleotidebis ramodenimejer ganmeorebadi 9-fuZe wyvilis (DnaA-box) da 13-fuZe wyvilis Semcveli specifiuri Tanamimdevroba. E.Coli baqteriaSi replikaciis iniciaciis mTavar komponents DnaA-cila warmoadgens, romelic oligomeris warmoqmnis Sedegad ATP-damokidebul reaqciaSi amoicnobs 13 fuZe wyvilian ganmeorebebs (AT-fuZe wyvilebiT mdidari ubani). DnaA-cilebis kompleqsi iwvevs dnm-is am monakveTze ormagi jaWvis ganxvevas da gancalkevebas [107-110]. amis Semdeg, DnaB cila DnaC-is daxmarebiT ukavSirdeba am ubans da iwyeba dnm-is ormagi spiralis orive mimarTulebiT gaSla. rac Seexeba eukariotul ujredebs, mag. adamianis qromosomis SemTxvevaSi, replikacia mimdinareobs mravali sawyisi wertilidan orive mimarTulebiT. cnobilia, rom DnaA cilis struqtura

28

homologiuria im cilebisa, romlebic arqi-baqteriebis da eukariotuli ujredis dnm-is replikaciis iniciator cilebs ganekuTvnebian [111,112], amitomac DnaA cilis struqturis da misi dnm-Tan kompleqsis Seswavla, rogorc modeli eukariotul ujredebSi mimdinare analogiuri procesebisa, aqtiurad mimdinareobs msoflios wamyvan laboratoriul centrebSi.

DnaA cila Sedgeba oTxi funqciurad damoukidebeli domenisagan [113,114]. III da IV domenebi amJRavneben ATP-aza (III domeni) da dnm-Tan specifiurad dakavSirebis (IV domeni) aqtivobas. es domenebi ufro konservatulebi arian, vidre I da III domenebi, romlebic rogorc Cans CarTuli arian oligomerizaciis da DnaB-Tan dakavSirebis procesSi. cnobilia, rom DnaA cila amoicnobs xuT 9-fuZe wyvilian Tanamimdevrobas OriC-Si, C-terminaluri spirali-maryuJi-spiralis (IV domeni) saSualebiT [115,116]. rac Seexeba N-terminalur domens, igi saWiroa DnaA cilebis kooperatiuli dakavSirebisaTvis mezobel DnaA box-ebTan. Catarebuli mutaciuri analizis mixedviT, cilis am ubanSi (I domeni) gansakuTrebiT mniSvnelovania Leu5, Trp6, Glu8 da Cys9 aminomJavas naSTebi [117,118]. t. louerisa da sxvebis mier [119,120] naCvenebia, rom DnaA cilis N-terminalur domenis struqturaSi arsebobs mJava aminomJavebis koncentrirebis ori ubani: Asp23 da Asp28, romelic mdebareobs hidrofobuli Raris zeda mxares, xolo cilis meore mxares ganlagebulia mJava aminomJavebis mTeli rigi Glu 46-dan Glu 67-mde III da IV spiralebis RerZis gaswvriv. Tumca maTi roli cilis funqciaSi dReisaTvis garkveuli ar aris.

dRemde arsebuli sam ganzomilebiani struqtura cila-dnm-is kompleqsisa aCvenebs, rom amomcnobi cilebis dnm-Tan kompleqsi Seicavs urTierTqmedebebis daxlarTul qsels, romelic akavSirebs partnior cilebs erTmaneTTan da dnm-Tan. individualuri urTierTqmedebebis wvlilis gansazRvra amocnobis procesSi sakmarisad rTulia. saboloo jamSi SegviZlia vTqvaT, rom nukleinis mJavasTan dakavSirebis specifiuroba Zlier unda iyos damokidebuli am mniSvnelovnad mcire gansxvavebaze kavSirebs Soris, anu im sust kavSirebze romelTa erToblioba warmoqmnis urTierTqmedebebis mtkice qsels [121].

29

II. Tavi.

eqsperimentuli nawili

2.1 sakvlevi masalebi

eqsperimentuli kvlevis obieqtad gamoyenebuli iyo xbos elenTidan

gamoyofili liofilizirebuli dnm-i 42 GC % wyvilebis SemcvelobiT,

sadac cilebis Semcveloba ar aRemateboda 0.1% da rnm-is koncentracia iyo

5% - ze naklebi (dnm-i mowodebuli iqsna prof. d. landos mier, risTvisac mas did madlobas vuxdi). dnm-is xsnaris dasamzadeblad analizuri sasworiT (ВЛР-20 sizuste~5·10

-5grami) xdeboda liofilizirebuli nimuSis awonva. dnm-is Temodinamikuri kvlevebisaTvis gamoyenebuli iyo ori tipis buferuli xsnari - mJave areSi SerCeuli iyo fosfat-limonmJavas buferi (0.02M Na2HPO4+0.01M limonmJavas buferi, pH 7.5), xolo tute areSi kvlevebisaTvis borismJava-tute buferi (0.02M H3BO3,+0.02M NaOH, pH 9.9). dnm-is sakvlevi nimuSis koncentraciis Semowmeba xdeboda speqtrofotometrulad, risTvisac viyenebdiT HEλIOS β, Thermospectronic

(Thermo Fisher Scientific, aSS) da Speqtromom 204 (ungrTi) speqtrofotometrebs. dnm-is koncentracia iTvleboda imis gaTvaliswineriT, rom 50 mkg/ml

koncentraciis dnm-is xsnaris STanTqma =260nm sinaTlis talRis sigrZis dros erT santimetris sisqis kiuvetaSi (eqstinciis koeficienti) Seadgens

STanTqmis erT erTeuls (260=1OD). sasurveli mJavianobis dnm-is xsnaris misaRebad gamoyenebuli iyo

wonasworuli dializis meTodi, raTa daculi yofiliyo gamxsnelis ionuri Zalis mudmivobis piroba, Termodinamikur parametrebze misi gavlenis gamosaricxad. rogorc tute, aseve mJave areebSi ganxorcielebul eqsperimentebSi ionuri Zalis mudmivoba miiRweoda buferis titrirebiT 1% HCl -is ganzavebuli xsnariT, iseve rogorc igive mJavis xsnars viyenebdiT tute buferis titrirebisas, sadac buferis pH-i dializis meTodis gamoyenebiT icvleboda maqsimaluri pH10-dan neitralur pH7 –mde mimarTulebiT.

dnm-Tan araspecifiuri urTierTqmedebis Sesaswavlad gamoyenebuli iyo PLGA (poly(D,L-lactitide-co-glycolic acid)) nanonawilakebi, romelic dResac intensiurad gamoiyeneba farmacevtebis mier rogorc wamlis gadamtani nawilakebi. eqsperimentebis mizani, garda araspecifiuri urTierTqmedebis Seswavlisa, iyo dagvedgina Tu ra sigrZis dnm-i SeiZleba iyos moTavsebuli nawilakis zedapirze (an SigniT) da SesaZlebelia Tu ara nawilakis meSveobiT moxdes saSualo zomis genis gadatana. rogorc avRniSneT eqsperimentebSi gamoyenebuli iyo PLGA-nanonawilakebi, romlebic damzadebuli iyo saarbrukenis universitetis farmaciis departamentSi poliglikolis mJavasa (PGA) da polilaqtidis mJavas (PLA) blokebisgan (70:30 procentuli TanafardobiT), romlebic Semdgom igzavneboda TviTmfrinaviT TbilisSi. eqsperimentebi tardeboda ori gansxvavebuli tipis (orive warmoadgens PLGA-nanonawilaks) xitosan-SaliTiani (cNp) da uSaliTo (Np) nanonawilakebze, romelTac gaaCniaT sferos forma zomebiT ~ 148 nm (sur. 12). gansxvaveba am nawilakebs Soris mdgomareobs zedapiruli muxtis sidideSi da niSanSi. kerZod, uSaliTo nanonawilakebis (Np) zedapiruli muxti iyo -8.6mV-i, xolo xitosan-SaliTianis (cNp) zedapiruli muxtis mniSvneloba Seadgenda +39.98mV-s.

30

sur. 12 PLGA-nanonawilakis suraTi

2.2. naSromSi gamoyenebuli meTodebis aRwera

2.2.1. centrifugirebis da speqtrofotometruli meTodebi

dnm- PLGA nanonawilakebis urTierTqmedebis kvlevebSi gamoyenebuli iqna ultracentrifugirebis meTodi. amisaTvis viyenebdiT Beckman CoulterTM,

AllegraTM

64R (aSS) firmis centrifugas, romlis maqsimaluri brunvis siCqare 25 000 br/wT-ia.

speqtrofotometruli kvlevebisTvis viyenebdiT HEλIOS β-s (Thermospectronic, Thermo Fisher Scientific, aSS), romlis gazomvis sizuste

Seadgens ±0.01 OD.

eqsperimentebSi gamoyenebuli iqna eleqtronuli mikro pH-metrebi (modelebi: HI98103 da HI98107; germania), romelTa gazomvis sizuste ±0.1 pH-is tolia.

31

2.2.2. diferencialuri mikrokalorimetruli meTodi

diferencialuri skanirebadi mikrokalorimetria gamoiyeneba

sxvadasxva tipis biopolimerTa SeswavlisaTvis, iseTebi rogorebicaa: globuluri da fibrilaruli cilebi, nukleinis mJavebi, fosfolipidebi biologiuri membranebi da sxva.

sxvadasxva faqtorebis (temperatura, pH, ionuri Zala da a.S) moqmedebis Sedegad biologiuri struqturebis daSlis procesis kalorimetruli kvleva ganpirobebulia imiT, rom es erTaderTi pirdapiri meTodia am procesebis energetikuli parametrebis gansasazRvravad. temperatura, rogorc biopolimerul struqturaze zemoqmedebis faqtori gansxvavdeba sxva faqtorebisagan imiT, rom is warmoadgens intensiur parametrs, romelic Termodinamikurad SeuRlebulia enTalpiasTan, romelic warmoadgens sistemis eqstensiur parametrs. es niSnavs, rom sistemis mdgomareobis yvela cvlilebas, romelic ganpirobebulia temperaturis zemoqmedebiT, Tan unda axldes enTalpiis cvlileba. ase rom, sistemis enTalpiasa da temperaturas Soris funqcionaluri damokidebuleba gulisxmobs Termodinamikur informacias im mdgomareobebze, romlebic dasaSvebia sistemisaTvis temperaturaTa ganxilul ubanSi [122,123].

temperaturaTa farTo intervalSi ბიომაკრომოლეკულების siTbotevadobis gasazomad dResdReobiT iyeneben siTbotevadur kalorimetrebs. arsebobs bevri gansxvavebuli modifikacia am xelsawyoebisa, romlebic gankuTvnilia sxvadasxva agregatul mdgomareobaSi nivTierebaTa siTbotevaduri kvlevebisaTvis sxvadasxva temperaturul intervalSi [124-126].

eqsperimentebs atareben molekulis ganzavebul xsnarebSi, ris gamoc SesaZlebelia molekulaTaSoris urTierTqmedebis ugulebelyofa, magram aseT xsnarebSi makromolekulebis mcire koncentraciis gamo gazomili siTburi efeqtebi sakmarisad mcirea. mcire siTburi efeqtebis sakmarisi sizustiT gansazRvra moiTxovda zepreciziul xelsawyos Seqmnas, rac SesaZlo gaxda mxolod diferencialuri adiabaturi mikrokalorimetris SeqmniT.

eqsperimentul kvlevebSi gamoyenebuli iyo rusuli warmoebis diferencialuri mikrokalorimetri ДАСМ-4A, romlis ZiriTadi teqnikuri monacemebi moyvanilia cxrilSi 3. am xelsawyos unikaluri mgrZnobiaroba miiRweuli iqna sami meToduri principis erTobliobiT: gazomvis diferencialuri sqemiT, uwyveti gaTbobiT (skanirebiT) zustad mocemuli siCqariT da gasazomi kamerebis sruli adiabatizaciiT.

kalorimetruli ampulebi gakeTebulia sruliad gamWoli kapilarebisagan, romlebic daxveulia spiralurad (sur. 13). ampulebis aseTi konstruqcia iZleva rig upiratesobebs: pirvel rigSi, gaadvilebulia maTi garecxva da avseba nimuSiT ise, rom am procedurebis Catareba ar saWiroebs maT daSlas da aseve iZleva SesaZleblobas ampulebze ganxorcieldes garkveuli wneva. raTa Casxmul siTxeSi ar moxdes haeris buStukebis gaCena; meore – kapilaris mcire diametris da misi maRali siTbogamtarobis gamo (platinis kapilari) ar xdeba siTxeSi temperaturis grantiebis warmoqmna, aqedan gamomdinare aseT kalorimetrul kameraSi temperaturaTa veli erTgvarovania da SeiZleba maTi gaxureba ufro didi siCqariT, vidre

32

moculobiTi (didi zomebis) ampulebis SemTxvevaSi. Tavis mxriv, skanirebis siCqaris gazrda niSnavs mowyobilobis realuri mgrZnobiarobis gazrdas.

rogorc avRniSneT kapilaruli ampulebi uZleben maRal wnevas da Casxmul xsnarebze arsebuli wneva (4-5 atmosfero) saSualebas iZleva gaizardos kalorimetris samuSao maqsimaluri temperatura 150

0C

niSnulamde, rac mniSvnelovnad zrdis bevr eqsperimentebis Catarebis SesaZleblobas.

ДАСМ-4A kalorimetris blok-sqema mocemulia sur. 14-ze, xolo kalorimetris teqnikuri monacemebi moyvanilia cxril 3-Si

cxrili 3

xmauris done 5 10-7

W

bazisuri xazis ganmeorebadoba 3 10-6

W

bazisuri xazis gadaxra swori xazidan 15 10-6

W

samuSao temperaturuli intervali 0 150С

temperaturis gazomvis absoluturi cdomileba 0.1 С

kalorimetruli ujrebis gacxelebis siCqare 0.125; 0.25; 0.1; 1.0; 2.0 ; 4.0 /min

skanirebis siCqaris fardobiTi cdomileba 2

wneva ampulis Tavze 500-600kpa

TiToeuli kameris moculoba 0.8 ml

samuSao moculoba 0.46 ml

kalibrebis simZlavre 25; 50; 100 W

33

sur. 13 DACM-4A tipis mikrokalorimetris kalorimetruli kamera:

1 _ platinis kapilarebi

2 _ uJangavi foladis Txeli milebi

3 _ Termobatarea

34

sur. 14 ДАСМ-4A tipis mikrokalorimetris blok-sqema:

1. monostati

2. Termometri

3. TviTmweri

4. siTburi efeqtis gamzomi mowyobiloba

5. Termostati

6. adiabatizirebuli garsis Termoregulatori mowyobiloba

7. mikrokalorimetris gamzomi kamerebi

35

2.2.3. mikrokalorimetruli mrudebis damuSaveba

nax. 3-ze mocemulia tipiuri kalorimetruli Canaweri, romelic daimzireba biopolimerebis (cilebi, nukleinis mJavebi da a.S) xsnarebis gacxelebisas, Tavisi bazisuri xaziTa (Canaweri skanirebisas gamxsneli-gamxsneli) da sakalibro (romelic Seesabameba gansazRvrul sakalibro energias) niSnuliT.

denaturaciuli pikis mier Semofargluri farTis mixedviT SeiZleba ganisazRvros biomakromolekuluri struqturis denaturaciis siTbo. amisaTvis SeiZleba vixelmZRvaneloT formuliT:

SKQ 1 (1)

sadac Q denaturaciis siTboa, S STanTqmis pikis qveS mdebare

farTia, K1 ki energetikul erTeulebSi Canaweris danayofis fasi (K1=Wett/Set, Wet- etalonuri simZlavre, romelic miewodeba erT-erT kameras t drois ganmavlobaSi, Set ki kalibruli niSnulis farTia).

siTbotevadobebis sxvaoba ampulebs Soris Cp nebismier temperaturaze SeiZleba ganisazRvros formuliT:

S

S

pp

p

pp mCmCC ][][ (2)

sadac [C]pp da [C]p

s - gaxsnili da gamxsneli nivTierebebis parcialuri siTbotevadobebia Sesabamisad, mpM-ampulis muSa moculobaSi

gaxsnili nivTierebis masa, ms–gaxsnili nivTierebis mier gamodevnili gamxsnelis masa. radganac

S

P

PSV

Vmm (3)

sadac [V]p da [V]

s aris parcialuri moculobebi nivTierebisa da gamxsnelisa Sesabamisad, amitomac sabolood vRebulobT:

p

p

S

pS

p

p

pm

C

V

VCC

][

][][][ (4)

sadac ampulebs Soris siTbotevadobebis sxvaoba Cp=k2h, sadac h aris wertilis gadaxra bazisuri xazidan nebismier temperaturaze, xolo

k2 aris gadaxris erTeulis fasi (santimetrebiT gazomili gadaxra wrfidan; ix. nax. 3 ), gamosaxuli j/K-Si;

k2= Wet / Let (5)

sadac Wet -kalibruli simZlavrea, -gaxurebis siCqare K/wm-Si, Let – ki kalibruli niSnulis gadaxra wrfidan.

sabolood, siTbotevadobis gamosaTvlelad viyenebdiT formulas:

p

S

pS

p

p

pmL

hW

V

VCC

][

][][][ (6)

36

nax. 3 biomakromolekulebis lRobis maxasiaTebeli tipiuri

kalorimetruli mrudi

37

eqsperimentalurad, sistemis enTalpiis temperaturaze damokidebuleba ganisazRvreba misi siTbotevadobis gazomviT. aRsaniSnavia, rom siTbotevadoba warmoadgens enTalpiis warmoebuls temperaturiT mudmivi wnevis pirobebSi (Cp=dH/dT). Tu cnobilia rogor icvleva sakvlevi makromolekulis parcialuri siTbotevadoba temperaturaze, SegviZlia ganvsazRvroT enTalpiaTa, entropiaTa da Tavisufal energiaTa sxvaoba makromolekulis natiur da denaturirebul mdgomareobaTa Soris gazomvis mTlian temperaturul intervalSi, radgan:

H(T0) = Td

To

C][ pndT+ Hd +

To

Td

C][ p ddT = Hd -

Td

To

CpdT ,

S(T0) = Td

To

{[ C]pn /T}dT + Hd /Td +

To

Td

C]{[ pd / T}dT = Hd /Td -

Td

To

{Cp /T }dT ,

G(T0) =H(T0) – T0S(T0) = Hd – T0{Hd/Td} –T0 Td

To

CpdT+ Td

To

{ Cp/T}dT =

Hd(Td-T0)/Td - Td

To

{( T-T0)/T}CpdT

Cp = [C]pd

–[C]pn aris gamxsnelSi gaxsnili molekulebis parcialur

siTbotevadobaTa sxvaoba denaturirebul da natiur mdgomareobebs Soris, xolo To da Td aris denaturaciis procesis dawyebis da lRobis

pikis Sesabamisi temperaturebi, xolo Hd - denaturaciis enTalpiaa. kalorimetruli mrudebis damuSaveba xorcieldeba specialurad

Seqmnili programiT [127] zemoT moyvanili (6) formulis gamoyenebiT, romelic gvaZlevs damokidebulebas sakvlevi obieqtis kuTrisiTbotevadobisa temperaturaze.

38

2.3. miRebuli eqsperimentaluri Sedegebi

nax. 4-ze naCvenebia dnm-is kuTri siTbotevadobis damokidebuleba

temperaturaze neitraluri pH7.0-is pirobebSi. naxazidan Cans, rom dnm-is denaturaciis mrudi xasiaTdeba e.w. lRobis nazi struqturiT (siTbosSTanTqmis piki Seicavs mcire pikebs (nax. 4)). unda avRniSnoT, rom miRebuli mrudis e.w. nazi struqtura, romelic ganpirobebulia dnm-is molekulaSi msgavsi Termostabilobis ubnebis (e.w. satelituri ubnebi) lRobis Sedegad, SesaZlebelia gamoyenebuli iyos imisaTvis, rom Cvens eqsperimentebSi ganisazRvros dnm-is renaturirebis xarisxi, rac Cvens eqsperimentebSi mniSvnelovania, rac gulisxmobs imas, rom Tu dnm-i denaturaciis mere mTlianad aRidgens Tavis sivrcul struqturas, maSin temperaturuli lRobis nazi struqturac iqneba damzerili. zogadad, SegviZlia vTqvaT, rom dnm-is kalorimetruli eqsperimentebidan miRebuli Termodinamikuri parametrebis mniSvnelobebiT da mrudis formiT SesaZlebelia vimsjeloT makromolekulis struqturis mowesrigebulobis xarisxze (mag. dnm-i spiralobis xarisxi) da mis Termostabilobaze, rasac warmodgenil naSromSi gadamwyveti mniSvneloba aqvs dnm-is struqturuli dinamizmis TvalsazrisiT.

nax. 5-ze mocemulia dnm-is kalorimetruli mrudebi mJave areSi, romlis mixedviT naTlad Cans, rom mJavianobis momatebiT (pH-is SemcirebiT) xdeba dnm-is lRobis temperaturis mkveTri Semcireba. amasTanave, temperaturis Semcirebis paralelurad mcirdeba dnm-is lRobis pikis farTic, anu enTalpiac. cxrili 4-Si mocemulia Catarebuli eqsperimentebis Sejamebuli monacemebi, romelic gviCvenebs, rom mJavianobis momatebiT Tu rogor icvleba dnm-is spirali-gorgali gadasvlis (denaturaciis) temperatura da enTalpia. miRebuli mrudebidan aseve kargad Cans, rom mJavianobis momatebiT dnm-is (pH 7.0) tipiuri kalorimetruli mrudisTvis damaxasiaTebeli patara pikebi (lRobis nazi struqtura) TandaTan qreba, rac SeiZleba imiT aixsnas, rom garemos garkveuli mJavianobis pirobebSi dnm imyofeba nawilobriv denaturirebul mdgomareobaSi, sadac SedarebiT naklebad Termostabilobiluri (AT-wyvilebiT mdidari) ubnebi ukve

gancalkevebulia, lRobisas STanTqmuli siTboc (H) Sesabamisad naklebia da aseT pirobebSi dnm-is lRobis nazi struqturac iwyebs gaqrobas (ix. nax. 5).

Catarebuli eqsperimentebidan Cans, rom mocemuli ionuri Zalis

pirobebSi, gamxsnelis pH 2.6 mniSvnelobamde dnm-is molekula inarCunebs or jaWvian spiralur ubnebs, rac Cans kalorimetrul mrudze siTbosSTanTqmis pikebis arsebobiT, xolo ufro dabali mJavianobis pirobebSi (pH≤2.6) dnm-i srulad denaturirebulia (nax. 6, II-mrudi) da siTbosSTanTqmis piki aRar daimzireba.

mJave aresagan gansxvavebiT dnm-is molekulas sruliad gansxvavebuli yofaqceva axasiaTebs tute areSi. Cvens mier Seswavlil iqna mudmivi ionuri Zalis pirobebSi dnm-is konformaciuli cvlilebebi tute areSi (pH 7.0-10.0). nax. 7-ze mocemulia dnm-is mikrokalorimetruli mrudebi, romlis mixedviTac tutianobis momatebiT (7.0<pH<10.0 intervali) adgili aqvs lRobis temperaturis (Tm) jer gazrdas da Semdeg TandaTan

Semcirebas, xolo rac Seexeba entlpiis cvlilebas (H) – igi monotonurad izrdeba

39

nax. 4 dnm-is kuTrisiTbotevadobis temperaturaze damokidebulebis

mikrokalorimetruli mrudi, 0.02M natriumis fosfati + 0.01M limonmJavas buferi pH 7.0, gacxelebis siCqare 2KK/min

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

8

Cp

[Jg

-1K

-1]

T [ 0C]

40

nax. 5 dnm-is kuTrisiTbotevadobis temperaturaze damokidebulebis

mrudebi garemos sxvadasxva mJavianobis pirobebSi (pH 7.0-2.8). gamxsneli _

0.02M natriumis fosfati+0.01M limonmJavas buferi

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

8

pH7.0

pH 4.6

pH 3.2

pH 3.0

pH 2.8

CP

[J

g-1K

-1]

T [ 0C]

41

nax. 6 dnm-is lRobis kalorimetruli Canawerebi. gamxsneli _ 0.02M natriumis fosfati+0.01M limonmJavas buferi. (I) -

dnm-is nimuSi pH 7.0 garemo pirobebSi, (II) - dnm-is nimuSi pH7.0-dan dializiT gadayvanili pH 2.6 pirobebSi, (III) - dnm-is nimuSi pH2.6 -dan dializiT dabrunebuli pH 7.0 pirobebSi

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

8

III

II

I

T [0C]

Cp [

Jg

-1K

-1]

42

nax. 7 dnm-is kuTrisiTbotevadobis temperaturaze damokidebulebis mrudebi tute garemo pirobebSi. gamxsneli _ 0.02M boris mJava+0.02M NaOH

buferi, I - pH 8.1, II - pH 8.6, III - pH 9.4, IV - pH 10.0.

20 40 60 80 1000

1

2

3

4

5

6

7

8

Cp

[J

g-1K

-1]

T [0C]

I

II

III

IV

43

nax. 8 dnm-is konformaciuli gadasvlis temperaturuli intervalis damokidebuleba garemos pH-ze (tute are)

7,5 8,0 8,5 9,0 9,5 10,0 10,510

11

12

13

14

15

16

T

0C

pH

44

(ix. nax. 7). tutianobis momatebisas, aseve SesamCnevi iyo dnm-is siTbos

STanTqmis pikis daviwroeba, anu lRobis temperaturuli intervalis (T) Semcireba (ix. nax. 8). miRebuli eqsperimentuli Sedegi metad sayuradReboa, ris Sesaxebac qvemoT (Semdeg TavSi) moyvanilia misi Cveneuli analizi.

nax. 9 da nax. 10-ze moyvanilia Sejamebuli dnm-is fazuri gadasvlis

temperaturis (Tm) da lRobis enTalpiis cvlilebis (H) damokidebulebis grafikebi gamxsnelis mJavianobis farTo intervalSi, romelic moicavs rogorc mJave ase tute ares - pH 2.8-10.0. eqsperimentuli Sedegebidan miRebuli Termodinamikuri parametrebis ricxviTi mniSvnelobebi moyvanilia cxrilSi (ix. cxrili 4).

zemoT moyvanili eqsperimentuli Sedegebis Tanaxmad sruliad naTelia, rom dnm-is mowesrigebuli struqtura mniSvnelovnad aris damokidebuli garemos mJavianobaze da misi cvlileba molekulaSi ganapirobebs mniSvnelovan struqturul cvlilebebs. bunebrivia, Cndeba kiTxva imis Sesaxeb, Tu ramdenad Seqcevadia aseTi struqturuli cvlilebebi dnm-Si, kerZod, kvlevebis mizani iyo dagvedgina mJave zemoqmedebiT detsabilizirebuli struqturis dnm-is molekulis mier sawyisi _ ormagspiraluri struqturis aRdgenis unari neitraluri pH 7.0 pirobebSi dabrunebisas. rogorc avRniSneT, pH-is cvlilebiT xdeba dnm-is struqturuli parametrebis cvlileba, romelic asaxulni arian miRebul Termodinamikur parametrebSi da mrudis formaSi, magram CvenTvis mniSvnelovani iyo dadgenili yofiliyo pH-is is minimaluri zRvari, romlis drosac dnm-is molekula neitraluri pH 7.0-is pirobebSi dabrunebisas mTlianad aRidgends Tavis pirvandel natiur ormagspiralur struqturas. nax. 11-ze mocemulia am eqsperimentis Sedegebi, sadac I - mrudi warmoadgens dnm-is siTbur lRobas pH 2.8

mJavianobis garemo pirobebSi, xolo II - mrudi – pH 2.8-dan wonasworuli dializis meTodiT neitraluri pH 7.0 garemoSi dabrunebuli dnm-is nimuSis kalorimetrul Canawers. rogorc grafikebidan Cans, mJave garemoTi (pH 2.8) destabilizirebuli dnm-is molekula neitralur pirobebSi dabrunebisas srulad aRidgens natiur ormag-spiralur struqturas, razec naTlad miuTiTebs, rogorc lRobis temperaturis da enTalpiis mniSvnelobebi aseve e.w. dnobis nazi struqturis arsebobac (ix. nax. 11-ze II-mrudi).

dnm-is mJava areSi destabilizaciisa da fiziologiur pirobebSi misi dabrunebisas dnm-is molekulaSi konformaciul cvlilebebze dakvirveba xdeboda aseve ultraiisferi speqtrofotometris saSualebiT. nax. 12-ze moyvanilia dnm-is STanTqmis speqtrebi sxvadasxva pH-is garemo pirobebisas, kerZod, pH 7.0 (uwyveti), pH 3.0 (wertilebi), pH 2.8 (wyvetili) saidanac Cans, rom fiziologiuri garemo pirobebidan dnm-is xsnaris mJave areSi gadayvanisas adgili aqvs speqtrebis wanacvlebas wiTel areSi. mniSvnelovania, rom Tu movaxdeT xsnaris mJave aredan wonasworuli dializis meTodiT neitralur pH 7.0 dabrunebas, dnm-is speqtri ubrundeba fiziologiur pirobebSi miRebul speqtrs (ix. nax. 13), romelic aseve miuTiTebs struqturis Seqcevad xasiaTze.

45

pH

lRobis temperatura

Tm 0C

0.1 0C

siTbos SnTanTqma

H J/g

5%

konformaciuli gadasvlis

temperaturuli intervali

T 0C

10%

2.8 16.0 9.0 _

3.0 22.0 17.4 12.3

3.2 28.0 20.6 12.3

4.6 60.0 44.2 14.7

5.7 69.0 47.2 13.5

6.6 71.0 46.7 13.7

7.0 71.3 47.3 14.1

7.5 71.6 47.8 13.5

8.1 74.3 49.0 14.7

8.6 74.5 53.6 14.1

9.0 73.3 54.8 12.5

9.4 71.0 56.0 13.5

10.0 68.5 58.4 11.8

cxrili 4 dnm-is Termodinamikuri parametrebi 0.02 M Na

+-is pirobebSi

46

nax. 9 dnm-is lRobis temperaturis garemos pH-ze damokidebuleba

nax. 10 dnm-is fazuri gadasvlisas enTalpiis cvlilebis garemos

pH-ze damokidebuleba

2 3 4 5 6 7 8 9 100

10

20

30

40

50

60

70

80

90

100

Tm

[ 0C

]

pH

47

nax. 11 mJavaTi destabilizirebuli dnm-is Seqcevadi xasiaTi misi

fiziologiur pirobebSi dabrunebisas. I _ dnm-is nimuSi 0.02M natriumis fosfati + 0.01M limonmJavas buferSi pH 2.8, II _ dnm-is nimuSi pH 2.8-dan wonasworuli dializis meTodiT gadayvanili neitraluri pH-is pirobebSi.

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

8C

p [J

g-1

K-1

]

T [ 0C]

I

II

48

nax. 12 dnm-is STanTqmis speqtri ultraiisfer ubanSi. wyvetili xazi _ dnm-is nimuSi pH 3.0 mJavianobis garemo pirobebSi.

wertilebi _ dnm-is nimuSi pH 2.8, uwyveti xazi _ dnm-is nimuSi pH 7.0

nax. 13 dnm-is STanTqmis speqtri ultraiisfer ubanSi. wyvetili xazi _ dnm-is nimuSi pH 2.8 mJavianobis garemo pirobebSi uwyveti xazi _ dnm-is nimuSi pH 2.8 -dan dializis meTodiT

gadayvanili neitraluri pH garemoSi

200 250 300 350 4000.0

0.2

0.4

0.6

0.8

1.0

1.2

Abso

rbti

on

/ nm

200 250 300 350 400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Abso

rpti

on

/nm

49

imisaTvis, rom dagvedgina warmoqmnis Tu ara PLGA nanonawilakebi

kompleqss dnm-Tan da gansazRvruli yofiliyo dnm-is (bazisuri wyvilebis) is raodenoba, romelic modis erT nanonawilakze, SevimuSaveT saimedo da swrafi meTodi. es meTodika moicavs speqtrofotometruli da centrifugirebis meTodebis kompleqsur gamoyenebas.

dakvirveba warmoebda dnm-is buferuli xsnaris STanTqmis speqtris cvlilebaze. Tavdapirvelad aRniSnul nimuSs vamatebdiT sufTa (uSaliTo) Np-PLGA-nanonawilakebis mcire porciebs (Np-PLGA/dnm-is maqsimaluri woniTi Tanafardoba 10/1), morevis Semdeg vacentrifugirebdiT 13 500 rpm siCqariT 10wT-is ganmavlobaSi, viRebdiT xsnaris supernatants (zeda xsnari) da xdeboda misi STanTqmis speqtris Cawera. rotoris aseTi siCqariT brunvis dros dnm-is sedimentacia ar xdeba, Tumca dnm-is sxva makromolekulebTan (PLGA nanonawilakebTan) kompleqsis warmoqmnis SemTxvevaSi dnm-is naleqi unda warmoqmniliyo, Sedegad ki zeda xsnarSi dnm-is koncentracia Semcirdeboda.

analogiuri eqsperimentuli meTodi iqna gamoyenebuli xitosan-SaliTiani cNp-PLGA nanonawilakebis dnm-Tan urTierTqmedebis Sesaswavlad, sadac nanonawilakebis porciebis damateba xdeboda safexureobrivad miRebiT: 1.685; 4.045; 6.726; 11.37. maqsimaluri raodenoba damatebuli cNp-PLGA-nanonawilakisa, romelic dakavSirebuli iyo dnm-Tan gansazRvrul iqna garkveul etapze centrifugirebis Sedegad miRebul supernatantSi dnm-is sawyisi STanTqmis speqtris sruli gaqrobiT.

Catarebuli speqtrofotometruli eqsperimentebis Sedegebi mocemulia nax. 14-ze da nax. 15-ze, sadac naCvenebia Np-uSaliTo (mcire uaryofiTi muxtis matarebeli) da cNp-xitosan-SaliTiani (dadebiTi muxtis matarebeli) PLGA-nanonawilakebis dnm-is damatebiT miRebuli xsnarebis dacentrifugirebis Sedegad miRebuli supernatantebis STanTqmis speqtrebi ultraiisfer areSi.

rogorc grafikebidan Cans, pirveli maTgani (cNp) aqtiurad urTierTqmedebs dnm-Tan da Sedegad warmoqmnili kompleqsis dacentrifugirebiT supernatantis STanTqma mniSvnelovnad mcirdeba. naTelia, rom xitosan-SaliTiani (cNp) PLGA-nanonawilakis dadebiTi zedapiruli muxti uzrunvelyofs dnm-is eleqtrouaryofiT fosfatur jgufebTan araspecifiuri, eleqtrostatikuri kavSirebis warmoqmnas (sur. 15). miRebuli eqsperimentuli Sedegebis (cxrili 5) mixedviT agebuli grafikis (ix. nax. 16) Tanaxmad cNp/dnm-is dakavSirebis maqsimaluri woniTi Tanafardobaa cNp/DNA=7:1 (w/w). rac Seexeba sufTa nanonawilaks (Np), rogorc Cans igi sustad an saerTod ar urTierTqmedebs dnm-Tan, ris gamoc centrifugirebis Sedegad dnm/Np-s supernatantis STanTqma TiTqmis ar icvleba (ix. nax. 14).

50

nax. 14 dnm-is buferuli xsnaris (1) da Np-dnm-is sistemis (2)

(Np/DNA 10:1) dacentrifugirebis Sedegad miRebuli supernatantis STanTqmis speqtri ultraiisfer areSi.

51

nax. 15 dnm-sa buferuli xsnaris (1) da cNp-dnm-is sistemis (2, 3, 4, 5)

(cNp/DNA Tanafardobebi _ 1.685; 4.045; 6.726; 11.37) dacentrifugirebis Sedegad miRebuli supernatantis STanTqmis speqtri ultraiisfer areSi

52

sur. 15 dnm-is ormagspiraluri polinukleotiduri jaWvis cNp-

PLGA-nanonawilakze daxvevis modeluri suraTi

53

nax. 16 cNp-PLGA/dnm-is (1) da Np-PLGA/dnm (2) sistemebis dacentrifugirebis

Sedegad miRebuli suparnatantebis STanTqmis (260nm) Sesabamis woniT Tanafardobebze damokidebulebis grafiki

cxrili 5

№

Np

(N

p)/

DN

A

w/w

A260

Np

cNp

1 0 2.00 2.00 2 1.7 1.93 1.53 3 4.0 1.93 1.00 4 6.7 1.92 0.10 5 9.0 1.92 0.05 6 11.4 1.92 0.02

54

2.4. miRebuli eqsperimentuli Sedegebis ganxilva

rogorc Catarebulma eqsperimentebma aCvenes, dnm-is fizikur

Tvisebebs mniSvnelovnad gansazRvravs gamxsnelis pH, rac asaxulia molekulis im Termodinamikur parametrebze damzeriT, romelic kalorimetruli eqsperimentebiT iyo dadgenili. Catarebuli eqsperimentebidan calsaxad Cans, rom mJave areSi (pH 7.0-pH 2.8) dnm-is struqtura ganicdis mniSvnelovan destabilizacias, maSin roca tute areSi molekula inarCunebs stabilur ormagspiralur struqturas (pH 7.0-pH 10.0). zogierTi literaturuli monacemebis Tanaxmad, rogorc zemoT avRniSneT Zlier mJave areSi (pH<4.6) xdeba dnm-is wyvilebis protonizacia, ris Sedegadac wyalbaduri kavSirebis ricxvi nukleotidur naSTebs, rogorc AT ise GC wyvilebs Soris erTiT mcirdeba [59]. Cvens mier miRebuli Sedegebi srul TanxvedraSia literaturaSi arsebul am monacemebTan, xolo damatebiT SeiZleba iTqvas, rom pH 2.6-is pirobebSi dnm-is molekulis jaWvebi erTmaneTisgan mTlianad arian dacilebulni, anu dnm srulad denaturirebulia.

mniSvnelovani Sedegia miRebuli dnm-is struqturuli labilobis Sesaxeb, rac imaSi gamoixateba rom molekulis Zlieri destabilizaciis miuxedavad (magaliTad pH 2.8-mde), jaWvebs Soris jer kidev SenarCunebulia wyalbaduri kavSirebi is minimaluri raodenoba, romelic sakmarisia raTa garemos pH-is am eqstremaluri mniSvnelobidan normalur, fiziologiur pirobebSi dabrunebisas molekulam srulad aRidginos natiuri, ormagspiraluri struqtura. gansakuTrebiT sayuradReboa is faqti, rom jaWvebs Soris kavSirebis aRdgena xdeba faqizi struqturebis donezec, razec miuTiTebs mJavaTi Zlier destabilizirebuli dnm-is neitraluri pH-is pirobebSi gadayvanisas misi siTbos STanTqmis mrudze patara pikebis kvlav gamoCena (nax. 11-ze II-mrudi).

rogorc eqsperimentebidan Cans sakmarisia dnm-is jaWvebi garemos

kritikuli mJavianobis gavleniT, kerZod pH2.6, mTlianad dacilebulni iyvnen erTmaneTisgan, rom SesaZlebloba imisa, rom komplementarul fuZeebs Soris kvlav moxdes kavSirebis sworad ganxorcieleba (ormagspiraluri struqturis aRdgena) aRar arsebobs. am SemTaxvevaSi

(pH2.6), molekulas ukve aRar aqvs unari misi fiziologiur garemo pirobebSi dabrunebis SemTxvevaSic ki daubrundes sawyis, natiur konformaciul mdgomareobas (nax. 6-ze III-mrudi).

saintereso Sedegia miRebuli dnm-is tute areSi kvlevebisasac. eqsperimentulad naCvenebia, rom tute areSi (pH 7.0-10.0), gansxvavebiT mJave aresagan, dnm-is molekula gamoirCeva maRali stabilobiT. eqsperimentulad miRebuli Canawerebidan naTlad Cans, rom tutianobis gazrdiT mcirdeba dnm-is konformaciuli gadasvlis temperaturuli intervali, anu dnm-is siTburi lRoba xdeba ufro kooperatiuli (nax. 7, ix. cxrili 4). amave dros, tutianobis gazrdiT pH7.0-10.0 intervalSi daimzireba denaturaciis STanTqmis enTalpiis ara Tu Semcireba aramed adgili aqvs mis monotonurad momatebasac ki (ix. nax. 10 da cxrili 4). literaturaSi arsebobs monacemebi [48] tute areSi dnm-is denaturaciis temperaturuli intervalis Semcirebis Sesaxeb, Tumca informacia lRobis enTalpiis mniSvnelobebis Sesaxeb (miTumetes mis gazrdaze) pH-is aRniSnul intervalSi literaturul wyaroebSi ar moiZieba. Cveni azriT miRebuli eqsperimentuli Sedegis mizezi (enTalpiis gazrdis tendencia) aris jaWvebs Soris arsebuli kavSirebis energiis gazrda, rac

55

TavisTavad metad sayuradRebo Sedegad SeiZleba CaiTvalos. miRebuli eqsperimentuli Sedegebis xasaiTidan gamomdinare erTerTi yvelaze misaRebi axsna SeiZleba iyos AT wyvilebs urTierTqmedebis gazrda ufro metad vidre GC wyvilebs Soris (mxolod Zlier tute ares pirobebSi). Tu am mosazrebas gaviTvaliswinebT, maSin SesaZlebelia aixsnas, rogorc dnm-is lRobis entalpiis gazrda aseve miRebuli siTbosSTanTqmis pikis daviwroebac. imisaTvis, rom gavrkveuliyaviT, Tu ramdenad marTebuli iyo Cveni mosazrebani, Catarebuli iqna Teoriuli gaTvlebi sistemisaTvis, romelic Sedgeboda adeninis, Timinis da NaOH molekulebisagan. aRniSnuli gamoTvlebi Catarebul iqna Tsu-s zust da sabunebismetyvelo mecnierebaTa fakultetis qimiis mimarTulebis profesorebis: jumber kereseliZis da Tamaz farcvanias mier. adenin-Timinis komplementarul wyvilze NaOH molekulis gavlenis raodenobrivi aRweris mizniT gamoyenebul iqna qvanturi qimiis Tanamedrove meTodi – simkvrivis funqcionalis Teoria (DFT) [128,129]. gamoTvlebi sruldeboda “Nature” programis mixedviT [130], PBE miaxloebis gamoyenebiT. garda amisa, Sedarebis mizniT gamoiyeneboda BLYP funqcionali [131,132] da lokaluri simkvrivis miaxloeba [133].

energiis optimizaciis Sesrulebuli gaTvlebis Sedegad dadgenil iqna, rom AT wyvilis da NaOH-is molekulis cal calke sruli energia tolia, Sesabamisad, 920.7917 da 237.9693 hartris (energiis atomuri erTeuli 1 hatri = 2625.5kj/mol). anu molekuluri sistemis jamuri energia aris E1= -1158.7610 hartri.

Semdeg gamoTvlili iqna AT wyvilis sruli energia NaOH-is Tanaobisas (gamoTvlebi kompiuterze mimdinareobda daaxloebiT 24 saaTis ganmavlobaSi), romelic aRmoCnda E2= -1158.7824 hartri.

Tu SevadarebT E1 da E2 energiebis mniSvnelobebs, vnaxavT, rom ukanasknelis Tavisufali energia aris Semcirebuli, anu molekuluri sistema mdgradia 56.2kj/moliT anu 13.4kkal/moliT:

-E= -1158.7842-(-1158.7610) = -0.0214 hartri= -56.2kj/mol= -13.4 kkal/mol. amave dros am gaTvlebiT iqna miRebuli, rom energiis Semcireba ganxorcielda AT wyvilebSi damatebiTi kavSiris gaCeniT, rasac Cveni eqsperimentuli SedegebiT vvaraudobdiT.

qvemoT moyvanilia AT da GC wyvilebs Soris dnm-Si wyalbaduri kavSirebiT warmoqmnili energiebis ricxviTi mniSvnelobebi. gamoTvlil iqna am sistemis sruli energia da cal calke A da T fuZeebis sruli energiebi:

Eh(A+T) = -453.8025 + (-466.9659) = -920.7684 hartri – cal-calke A da T

nukleotidebis energiebis jami. Eh(AT)= -920.7914 hartri - wyalbaduri bmebiT dakavSirebuli A da T

nukleotiduri wyvilis energia. maSin, E(HB)= E(AT)-E(A+T) = -920.7914-(-920.7684) = -0.023 hartri = -60.4kj/mol =

-14.4kkal/mol romelic Seesabameba AT wyvils Soris ganxorcielebuli

wyalbaduri kavSirebis energias. analogiuri midgomiT gamoTvlil iqna sami wyalbaduri bmis

energiis sidide GC wyvilebSi: Eh(G+C) = -542.1648+(-394.6416) = -936.8064 hartri – cal-calke G da C

nukleotidebis energiebis jami. Eh(GC) = -936.8500 hartri – GC kompleqsis energia

56

Eh(HB)= E(GC)-E(G+C)= -936.8500-(-936.8064)= - 0.0436 hartri = -114.6 kj/mol = -27.4kkal/mol, romelic warmoadgens GC wyvils Soris ganxorcielebuli wyalbaduri kavSirebis energias.

sur. 17-ze naCvenebia zemoT moyvanili gaTvlebiT programis mier dafiqsirebuli NaOH molekulis meSveobiT AT wyvilebs Soris damatebiTi kavSiris warmoqmnis adgili.

amrigad, Tu daveyrdnobiT zemoT moyvanil eqsperimentul Sedegebs, maTi analizis safuZvelze SeiZleba gakeTdes zogierTi Sefaseba imis Sesaxeb, Tu rogor SeiZleba gamoyenebul iqnas mJave areSi dnm-is molekulis struqturuli SesaZleblobebi ujredSi misi funqcionirebis TvalsazrisiT.

miRebuli eqsperimentuli Sedegebi miuTiTebs imaze, rom mJave garemo ujredma SeiZleba “gamoiyenos” imisTvis, rom molekulis jaWvebis gaxsna ganxorcieldes mcire energiis danaxarjiT. logikuria, rom Tu SesaZlebelia ujredSi dnm-is funqciuri ubnis (genis) gaxsnaSi daixarjos mcire energia, ujredi aseT SesaZleblobas aucileblad gamoiyenebs, miTumetes, rogorc miRebuli eqsperimentuli Sedegebi (mJavaTi destabilizirebuli dnm-is unari mTlianad aRidginos sawyisi ormagspiraluri struqtura) cxadyobs, rom aseT SemTxvevaSi dnm-is jaWvebis gaxsna/daxurva unda iyos sruliad Seqcevadi procesi. amas daemateba is garemoebac, rom literaturuli monacemebis Tanaxmad cilebi, romlebic CarTulni arian dnm-is funqcionirebis procesSi gamoirCevian Warbi raodenobis mJave bunebis aminomJavebis SemcvelobiT [134-146], rac Cveni azriT ar unda iyos SemTxveviTi. kerZod gamovTqvamT mosazrebas, rom is cilebi, romlebic mniSvnelovan rols asruleben genebis aqtivaciaSi, risTvisac isini warmoqmnian specifiur kompleqss dnm-is molekulis garkveul nukleotidur TanamimdevrobebTan, TavianT struqturaSi unda moicavdnen iseT ubans, sadac Tavmoyrili iqneba cilaSi Semavali mJave bunebis aminomJavebi, romelic moaxdens dnm-is ubnis (promotoruli ubani) destabilizacias an mTlianad jaWvebis lokalur, droebiT, gancalkevebas. am midgomiT SeiZleba gakeTdes garkveuli Sefasebac, romelic imaSi mdgomareobs, rom cilebi, romlebic cnoben dnm-is specifiur ubnebs da warmoqmnian kompleqss masTan, unda xasiaTdebodnen ori saerTo TvisebiT: am cilis SemadgenlobaSi unda Sediodes garkveuli (SedarebiT Warbi) raodenoba mJava bunebis aminomJavebisa da cilis mesameuli struqtura unda uzrunvelyofdes maTi Warbi raodenobis garkveul ubanSi Tavmoyras.

mniSvnelovani iyo PLGA-nanonawilakebis, rogorc genis gadamtani nawilakebis dnm-Tan urTierTqmedebis Seswavla, romelsac didi praqtikuli mniSvneloba aqvs farmakologiasa da genur TerapiaSi. xitosan-SaliTiani PLGA-nanonawlakisa da dnm-is araspecifiuri urTierTqmedebis speqtrofotometrulma kvlevebma cxadyo maTi stabiluri kompleqsis warmoqmnis SesaZlebloba. amasTanave agebul iqna cNp-PLGA/dnm-is sistemis centrifugirebis Sedegad miRebuli suparnatantebis STanTqmis Sesabamis woniT Tanafardobebze damokidebulebis grafiki (ix. nax. 16), romlis Tanaxmadac TiTo cNp-PLGA nawilaks SeuZlia miierTos dnm woniTi TanafardobiT 7:1 (cNp-PLGA:DNA,

w/w). Tu gaviTvaliswinebT nanonawilakis masas, romelic Seadgens M~3·10-

15g -s da dnm-is TiTo fuZe wyvilis molur masas (~700Da), maSin SegviZlia

viangariSoT Tu ra raodenobis fuZe wyvilis mierTeba (da gadatana) SeuZlia aseTi tipis erT nawilaks. es ricxvi aRmoCnda 3.68·10

5-is toli, rac savsebiT sakmarisia samizne ujredSi saWiro genis gadasatanad

57

(saSualod geni Seicavs 10-15 aTasamde fuZe wyvils [147]). gansxvavebulia suraTi uSaliTo Np-PLGA anononawilakis SemTxvevaSi, sadac araviTari urTierTqmedeba dnm-Tan ar dafiqsirda, rac advilad SeiZleba aixnas misi uaryofiTi zedapiruli muxtis arsebobiT, romelic gamoiwvevda ganzidvas dnm-is uaryofiTi muxtis matarebel fosfatur jgufebTan urTierTqmedebisas. amrigad, gamoikveTa xitosan-SaliTiani PLGA-nanonawilakis, rogorc genis gadamtani nawilakis udavo upiratesoba sufTa saxis PLGA-nanonawilakTan SedarebiT.

58

sur. 17 Teoriuli gaTvlebis Sedegad AT wyvilebs Soris

damatebiTi kavSiris warmoqmnis adgili.

59

daskvnebi 1. dadgenilia garemos mJavianobis gavlena dnm-is mowesrigebuli struqturis energetikul parametrebze, romlis mixedviTac dnm-is struqturuli labiloba da misi stabiloba mniSvnelovnad ganisazRvreba garemos pH-is mniSvnelobiT. naCvenebia, rom dnm-is Termodinamikuri Tvisebebi mJave garemoSi (pH 2.8-7.0), gansxvavebiT tute garemosgan (pH 7.0-

10.0), mniSvnelovnad aris damokidebuli gamxsnelis mJavianobaze. rac ufro maRalia garemos mJavianobis xarisxi, miT ufro naklebia dnm-is denaturaciis temperatura da STanTqmuli kuTri siTbo (entalpiis cvlileba). 2. naCvenebia (mocemuli ionuri Zalis pirobebSi) dnm-is sruli denaturaciisTvis saWiro garemos pH-is kritikuli mniSvnelobebi – mJave areSi es mniSvneloba Seadgens pH 2.6-s da tute aresTvis pH12.0 -s.

3. naCvenebia, rom dnm-is struqturis mJave garemoTi destabilizaciis procesi aris sruliad Seqcevadi pH 2.8 mniSvnelobidan dawyebuli, xolo pH 2.6 pirobebSi gadayvanisas dnm Seuqcevadad denaturirdeba. 4. naCvenebia, rom tute garemoSi (pH7.0-10.0) dnm-is lRobis temperaturuli intervali (pikis naxevarsimaRlis niSnulze) tutianobis momatebiT iwyebs Semcirebas, izrdeba denaturaciis STanTqmis siTbo (kuTri entalpia) da gadasvlis temperatura (pH 9.0-mde). miRebuli Sedegi aSkarad mianiSnebs AT da GC fuZe wyvilebis Termuli stabilobis daaxlovebaze da saboloo jamSi, dnm-is stabilobis gazrdaze. lRobis enTalpiis gazrda ganpirobebuli unda iyos AT wyvilebs Soris damatebiTi kavSiris warmoqmniT, rasac adasturebs Teoriuli gaTvlebi NaOH-is garemoSi AT wyvilebs Soris damatebiT mesame kavSiris warmoqmnis SesaZleblobis Sesaxeb, rac sruliad axali Sedegia. 5. miRebuli eqsperimentuli Sedegebis safuZvelze Camoyalibebulia modeli, romlis mixedviT cilebis mier dnm-is specifiuri ubnebis amocnobaSi mniSvnelovan rols unda TamaSobdes cilis struqturaSi arsebuli mJave aminomJaveebiT mdidari ubnebi. kerZod, cila-dnm-is specifiuri kompleqsis warmoqmnas ganapirobebs cilis mJava ubnis mier dnm-is am monakveTis (SedarebiT naklebad stabiluri AT-wyvilebiT mdidari ubani) despiralizacia (jaWvebis gancalkeveba), ris Semdegac adgili aqvs dnm-Tan cilis ufro mWidro urTierTqmedebas da e.w. Caketili kompleqsis warmoqmnas. 6. ori tipis (uSaliTo da xitosan-SaliTiani) PLGA nanonawilakis dnm-Tan urTierTqmedebis Seswavlis safuZvelze naCvenebia, rom mcire uaryofiTi muxtis mqone uSaliTo PLGA nanonawilaki ar urTierTqmedebs dnm-is ormagspiralur struqturasTan, maSin roca dadebiTi muxtis matarebeli xitosan-SaliTiani nawilaki sakmaod intensiurad ikavSirebs

mas, kerZod, 1 nanonawilaks unari aqvs miierTos 3.68·105 cali bazisuri wyvili, rac sruliad sakmarisia ujredSi erTi genis gadasatanad.

60

aRniSnul sadisertacio Temaze momzadebul iqna samecniero statiebi, romlebic dasabeWdad miRebul iqna Semdeg JurnalebSi:

1. M. Khvedelidze, T. Mdzinarashvili, T.Partskhaldze, N. nafee, U.F. Schaefer, C-M. Lehr, M.

Schneider, Calorimetric and spectrophotometric investigation of PLGA nanoparticles and their

comlex with DNA, J. Thermal Analysis and Calorimetry, 2009. (in press)

DOI: 10.1007/s10973-009-0137-x

2. T. Mdzinarashvili, T. Partskhaladze, M. Khvedelidze,T. Lomidze, Possible role of acidic

environment for the structural and functional abilities of DNA, 2009, J. Boil. Phys. Chem. Vol. 9,

pp.77-82.

3. T. Mdzinarashvili, M. Khvedelidze, T. Partskhaladze, N. Nafee, U. F. Schaefer, C.-M. Lehr,

M. Schneider, Biophysical approach to evaluate DNA transfer by PLGA nanoparticles, 2009, J.

Boil. Phys. Chem. Vol. 9, pp. 83-87.

4. "European Human Genetics Conference 2008", ( Barcelona, Spain)

T. Partskhaladze, T.Mdzinarashvili, T. Lomidze, "The acidic environment as essential attribute

for the DNA functioning" (Abstract), Journal of European Human Genetics, Vol. 16 supplement,

May 2008, p. 400.

61

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68

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