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Hoe bepaal die volgorde van die pare kruisbindings in DNS die rangskikking van die aminosure?

Hoe bepaal die volgorde van die pare kruisbindings in DNS die rangskikking van die aminosure?


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Deur Richard Feynman aan te haal uit hoofstuk 3 van sy boek Six Easy Pieces, wanneer hy oor DNA praat:

Geheg aan elke suiker langs die lyn, en verbind die twee kettings saam, is sekere dele van kruisbindings. Hulle is egter nie almal van dieselfde soort nie; daar is vier soorte, genoem adenien, timien, sitosien en guanien, maar kom ons noem hulle A, B, C en D.

Vervolgens kom die vraag, presies hoe bepaal die volgorde van die A, B, C, D eenhede die rangskikking van die aminosure in die proteïen? Dit is vandag die sentrale onopgeloste probleem in biologie.

Feynman het sy lesings gedurende 1961-1963 aangebied. Weet ons nou hoe die volgorde van adenien, timien, sitosien en guanien die volgorde van die aminosure in die DNA beïnvloed?


Ja, dit is opgelos in die vroeë 1960's, begin omstreeks 1961. Sien Wikipedia se Genetiese Kode - Geskiedenis, en dalk "Establishing the Triplet Nature of the Genetic Code".


Kruiskoppeling in DNA

  • Bygedra deur Ed Vitz, John W. Moore, Justin Shorb, Xavier Prat-Resina, Tim Wendorff en Adam Hahn
  • ChemPRIME by Chemical Education Digital Library (ChemEd DL)

DNA is een van die mees produktiewe biomolekules in terme van doel en replikasie. Sy gene stuur instruksies na RNA, aminosure, en gevolglik byna alle biomolekules uit om spesifieke eienskappe op te stel. Wanneer "unzipped", reageer stringe DNA met ekstra nukleotiede om twee identiese dubbelpaar stringe te produseer, 'n proses wat ontgin word in die Polimerase Kettingreaksie, waar 'n sekere volgorde van DNS geamplifiseer word deur gebruik te maak van grensinleiers en basisbou-polimerase.

Soms kan dit egter nuttig wees om die replikasie van DNA te stop, en 'n spesifieke monster vir ontleding te bewaar. Wetenskaplikes stel daarin belang om die menslike genoom te karteer en te bepaal waar spesifieke kernproteïene met die DNS-ketting in wisselwerking tree [1] . Om te verhoed dat DNS repliseer, kan stringe wees kruis gekoppel, of kovalent gebind, met sekere molekules. Die netwerk van kruisbindings verhoed in wese dat die DNS verander, maar veroorsaak ook dat dit na 'n tyd doodgaan. As gevolg hiervan behoort die kruiskoppelingsproses maklik omkeerbaar te wees om die DNA-molekule te laat oorleef.

Kruisbindingsreagense moet ook stabiliteit toon onder biomolekulêre analise en in staat wees om koppeling in 'n relatief presiese area te lokaliseer. Een algemene kruisbindingsmiddel is formaldehied, die eenvoudigste aldehied. Formaldehied se klein grootte en duursaamheid onder analitiese toestande wat nie normaalweg in die menslike liggaam voorkom nie, maak dit 'n ideale kandidaat [2] . Boonop maak inkubasie by 70 o C die kruisbinding ongedaan. Groter aldehiede in tabakrook en motoruitlaat kan soortgelyke maar onomkeerbare effekte hê, wat DNS permanent beskadig.

'n Voorbeeld van waar formaldehied nuttig kan wees in kruisbinding is die proses van chromatien-immunopresipitasie. Hierdie proses ontleed chromatien, wat die kombinasie van DNA en proteïene is wat chromosome uitmaak. Deur die liggings van sekere molekules bekend as histone te karteer, kan wetenskaplikes 'n rowwe kaart van proteïene in die genoom bou. Eerstens moet die proteïene en DNA egter in plek gehou word. Die byvoeging van formaldehied stel proteïen-proteïen- en proteïen-DNS-interaksies vas, wat ontleders in staat stel om die genoom te ondersoek en dit dan uitmekaar te verdeel met spesifieke teenliggaampies en sonikasie, in die immunopresipitasieproses. Dit isoleer kruisgekoppelde dele van DNA van gewenste ongebonde dele, wat wetenskaplikes kan versterk deur middel van PCR [3].

In sommige gevalle wil wetenskaplikes dalk die uitwerking op DNA waarneem wanneer hierdie bakterieselle geoksideer word. Te veel van 'n oksideermiddel, soos H2O2 sal die DNS doodmaak, maar te min sal nie die reaksie wat ons wil hê onwettig maak nie. Om dit reg te stel, kan kruisbinding gebruik word om die DNA teen 'n hoë vlak van oksidasie te beskerm. Disulfiedbindings, gevorm deur twee swaelmolekules, is algemene bindings wat gemaak word om hierdie effek te bereik.

Jy kan in die bostaande beeld van die proteïen sisten sien dat die individuele molekules elke swaelatoombindings identies lyk. Trouens, hulle is, en word sisteïenmolekules genoem, die disulfiedbinding verenig twee momomere om 'n dimeer, sistien, te skep. Disulfiedbindings en ander kruisbindingsmiddels is algemene reagense wat dimere en polimere uit individuele eenhede produseer.

In proteïene soos sistien kan die byvoeging van 'n sulfiedbinding nie net monomere aan mekaar koppel nie, maar hele lengtes proteïen om sekondêre proteïenstruktuur te vorm, soos beta-velle. Sulfiedbindings is van kritieke belang om te voorkom dat polêre watermolekules die amiedbindings uitmekaar breek.

Voorbeeld (PageIndex<1>): Bindingslengte

Gebaseer op wat jy weet oor tendense in atoomgroottes, bepaal watter binding langer is: 'n Disulfiedbinding of 'n C-C-binding. Hieruit, skat watter binding sterker is en of 'n koolstofruggraat- of disulfiedbinding meer geneig is om eerste te breek.

Atoomradiusse wat in die periodieke tabel afbeweeg as gevolg van 'n toenemende aantal orbitale, dus sal die binding tussen twee swaelatome langer wees as een tussen twee koolstofatome. Dit beteken die disulfiedbinding sal ook swakker as 'n C-C-binding wees, en sal waarskynlik eerste breek.

Disulfiedbindings is ook meer polariseerbaar as koolstof-koolstofbindings, as gevolg van 'n groter grootte en aantal elektrone. Elektrofiele soos die halogene aanvaar maklik die elektronpare op die swaelatome en breek die binding uitmekaar. 'n Algemene elektrofiel is isotiosianaat, met funksionele groep N=C=O [4] . Jy sal dalk sien hoe die koolstofatoom 'n binding met die naburige stikstof- en suurstofatome kan breek om twee nuwe enkelbindings met beskikbare elektronpare te vorm, soos dié op die tweewaardige swaelatome.

Disulfiedbindings kan 'n belangrike rol speel in die stabilisering van die tersiêre struktuur van proteïene, wat die entropie van die driedimensionele struktuur verlaag.


Lesingnotas oor die genetiese kode

Uit die studie van proteïensintese is dit duidelik dat aminosuurvolgorde in die proteïenmolekule bepaal word deur DNA wat inligting vir hierdie doel dra.

Maar waar lê die inligting? As ons na DNA-ketting kyk, blyk die deoksi-ribonukleotiede-ruggraat geen hulp te wees nie, aangesien dit 'n struktuur IS met herhalende nukleotiede, AG CT AT GC ensovoorts. Hier word die DNS-basisse adenien, guanien, sitosien en timien onderskeidelik met standaardletters A, G, C en T aangedui.

Die kode lê slegs in die vier basisse (A,T, G en C) wat baie keer in die ketting voorkom wat verskillende basisvolgordes aan verskillende DNA-stringe verskaf. Eintlik hou die 4 basisse in DNA-molekules in 'n gekodeerde vorm die boodskap vir die struktuur van proteïenmolekules.

Deur die transkripsieproses word die inligting wat in DNA gekodeer is na wRNA-band oorgedra. Dus word die ‘vierletter’ (vier basisse—A, T, G en C) taal van DNS getranskribeer na nog 'n komplementêre vierlettertaal in boodskapper-RNA.

Die volgende belangrike stap is die werklike vertaling van vierlettertaal van boodskapper-RNA in spesifieke proteïen met 20 letter-(aminosure) taal.

Aangesien die geen betrokke is by die sintese van proteïen en aangesien proteïen in sy primêre struktuur lineêre kombinasies van 20 aminosure verteenwoordig, is dit logies om af te lei dat die gekodeerde boodskap van die geen in die vorm van woorde moet wees wat die volgorde van bepaalde aminosure. Dit is wat ons bedoel met genetiese kode.

Maar hoe is dit dat net vier basisse in DNS die verskillende aminosure in die proteïene kodifiseer? As elke basis aan een aminosuur spesifiseer, kan slegs 4 aminosure gekodifiseer word. Indien twee aangrensende basisse (d.w.s. tweeletterwoorde) gebruik word, sal die maksimum aantal basispare (woorde) 4 x 4 = 16 wees wat slegs 16 verskillende aminosure kan uitmaak.

Maar die aantal verskillende aminosure is 20 waarvoor ten minste 20 verskillende woorde nodig sal wees. So tweeletterkodes los nie die doel op nie. As ons nou drieletterwoorde of drieletterkodes oorweeg (wat drie aangrensende nukleotiede insluit), dan sal 4x4x4 =64 verskillende tipes van drie laasgenoemde kodewoorde moontlik wees.

Hulle kan verantwoordelik wees vir 64 verskillende aminosure en dit is baie meer as 20 nodig. Die verskillende tipes moontlike mRNA-kodewoorde in elke geval is in Tabel 21.1 getoon.

Kenmerke van die genetiese kode:

1. In mRNA staan ​​die groep nukleotiede wat een aminosuur spesifiseer bekend as 'n kodewoord of “kodon”. Die boodskap word gelees as groep drie nukleotiede op 'n slag. Kode is kommavry.

2. Aangesien in drielingkodering die aantal kodons meer is as die aantal aminosure, word die hipotese veronderstel dat die genetiese kode gedegenereer sal wees. Gedegenereerde kodering beteken dat daar vir sommige aminosure meer as een kodon sal wees. Dit beteken dat 'n boodskapper-RNA, met twee uitsonderings (AUG en UGG), voorsiening kan maak vir die inkorporering van 'n bepaalde aminosuur meer as een drieling.

Benewens dit, sou 'n mens verwag om meer as een oordrag-RNA vir 'n aminosuur te vind, wat gedegenereerd gekodeer is. Onlangse navorsing het bewys dat hierdie verwagtinge korrek is. Kom ons kyk hier na 'n segment van 'n enkele ketting van DNA-molekule wat nege basisse (ATAGTCGAT) bevat.

As daar veronderstel word dat die genetiese kode kommaloos is, dit wil sê sonder spasieerwoorde (nie-sinwoorde) tussen sinwoorde, dan sal die gekodeerde boodskap op die volgende twee maniere gelees word:

1, As oorvleuelende woorde (Fig. 21.1).

2. As nie-oorvleuelende woorde (Fig. 21.1).

As die woorde op die oorvleuelende wyse gelees word, verskyn die derde nukleotied van DNA (dws. A) herhaaldelik in drie drieling ATA, TAG en AGT (Fig. 21.1), maar as die kode op nie-oorvleuelende wyse gelees word, dan derde nukleotied (A) verskyn slegs in een kode ATA.

Nou ontstaan ​​die vraag of die genetiese kode van oorvleuelende tipe of nie-oorvleuelende tipe is? Die eksperimentele bewyse is ten gunste van nie-oorvleuelende kode, dit wil sê, die aangrensende kodons oorvleuel nie.

Die argument is teen die konsep van 'n oorvleuelende kodesisteem gebaseer op die feit dat 'n verandering in derde nukleotied van DNA veranderinge in die drie aminosure in die oorvleuelende toestand sal meebring, want al die eerste drie trinukleotiede ATA, AG en AGT sal verander word deur 'n ander nukleotied as A in die derde plek te vervang.

Die gevolg sal wees dat proteïenmolekule wat in die veranderde toestand gevorm word, sal verskil van die normaal gevormde proteïen in drie aminosure. In die nie-oorvleuelende kode, aangesien slegs een woord verander sal word deur die vervanging van 'n nukleotied anders as A in die DNA-molekule op die derde plek, sal die proteïen wat in die veranderde toestand gevorm word, verskil van die wat in normale toestand in slegs enkele amino gesintetiseer word. suur.

Laasgenoemde toestand is werklik prakties gevind. Dit dui daarop dat 'n nie-oorvleuelende sisteem by genetiese kodering betrokke is. Die genetiese kode is universeel, dit wil sê, alle lewende organismes het dieselfde genetiese taal. Dit beteken dat 'n boodskap van 'n diersel dieselfde proteïen sal produseer of dit nou vertaal word deur proteïensintese-masjinerie van 'n bakteriese sel of plantsel.

4. Die genetiese kode gebruik spesifieke inisiasiekodon en stopkodons. AUG-drieling kodes vir metionien en is die inisiasiesein en as AUG afwesig is van die 5′ einde van mRNA, sal dit nie in 'n posisie wees om translasie of proteïensintese uit te voer nie. Van die 64 drielingkodes is drie stopseine of beëindigingseine of ‘non-sense’ kodons, UAG, UAA en UGA, wat die sintese van polipeptied stop.

5. Wobble Basisparing:

Dit is gevind uit volgorde-analise dat die paring van basis aan die 5′ einde van antikodon (dit is komplementêr tot derde basis van kodon) nie so streng of presies is soos die eerste en die tweede basisse nie en dit pare met meer as een basis aan die 3′ einde van die kodon i. e., U in die derde posisie van antikodon kan met A of G in kodon paar, G kan met C of U en I (inosiensuur met die basis hipoksantien) kan met U, C, A paar.

Dit word wankelbasisparing deur Crick (1996) genoem en slegs sekere parings is in hierdie verband moontlik. Wobble laat nie toe dat enige enkele /RNA vier verskillende kodons herken nie (Fig. 21.2).

Ontsyfering van genetiese kode:

As die genetiese boodskap eintlik in mRNA in die vorm van groepe van 3-letter kodewoorde vervat is, kan daar aanvaar word dat drie opeenvolgende basisse van boodskapper-RNA verantwoordelik sal wees vir die aanhegting van een aminosuur. Eksperimentele bewyse wat die konsep van drielingkode ondersteun, is deur F.H.C. Crick en medewerkers (1961).

Maarskalk Nirenberg en sy kollega John Matthaei was eerste om die genetiese kode in vitro-studie van proteïensintese te verbreek. Gewoonlik word radioaktiewe aminosure gebruik. Nirenberg en Matthaei het die ribosome, ensieme en energiebevrydende verbindings in 'n proefbuis geneem en dit versadig met die tRNA's wat verkry is uit die selekstrak van 'n bakterie Escherichia coli.

So het hulle die proteïensintetiseringsmeganisme van 'n sel in die proefbuis gestimuleer. In die eksperiment het hulle waargeneem dat die inkorporering van die aminosuur fenielalanien in proteïen aansienlik gestimuleer is deur die byvoeging van 'n kunsmatige RNA wat slegs uit urasielbasis bestaan ​​(d.w.s. mRNA wat UUUUUUUUUUUUU of poli U bevat).

Hierdie kunsmatige RNA-polimeer en soortgelyke ander polimere word gesintetiseer met behulp van 'n ensiempolinukleotiedfosforilase wat deur Ochoa, Nobelpryswenner, ontdek is. Aangesien poli-U-RNA-ketting slegs een basis-U bevat, is dit duidelik dat UUU die kode vir aminosuur-fenielalanien is, indien die drievoudige kodering korrek is. Dit het nuwe moontlikheid oopgemaak om die genetiese kode te breek.

Na hierdie belangrike ontdekking het Nirenberg, Ochoa en hul medewerkers die taak onderneem om kodons vir verskeie ander aminosure met RNA-polimere te vind. Hierdie RNA polimere het bestaan ​​uit óf een tipe basis soos poli U óf twee basisse, as poli AU(-AU-AU-AU ensovoorts) of drie tipes basisse, bv. poli AUG (AUC-AUC-AUC……………̵ 8230…………. en so aan). In sommige gevalle is die kodons vir aminosure maklik geïdentifiseer.

Bindingstegniek om genetiese kode te breek:

In baie gevalle was daar egter onduidelikheid aangesien die basisvolgorde in die kunsmatig gesintetiseerde boodskapper-RNA nie vooraf bepaal kon word nie. Verder was dit moeilik om die volgorde van basisse in 'n kodon te bepaal, bv. deur RNA-polimeer van drie basisse-A, U en C te maak, is daar ses moontlike maniere vir die rangskikking van basisse in drieling AUG, ACU, CAU, UGA en UAG.

Dit was nie maklik om te bepaal watter een kodon vir watter aminosuur geldig was nie. Antwoorde is deels verskaf deur die ontdekking wat Nirenberg en Leder (1964) gemaak het. Hulle het ontdek dat spesifieke tRNA-molekules aan ribosomale mRNA kan bind, selfs al is die mRNA saamgestel uit 'n enkele trinukleotied. Die binding van rRNA aan mRNA is nie moontlik as die mRNA slegs uit mono- of dinukleotiede bestaan ​​nie.

Die genetiese kode soos vasgestel vir die bakterie Escherichia coli (C.I. = kettinginisiasie C.T. = kettingterminasie of NONS = Nie-sin

A = Adenien, G = Guanien, C = Sitosien, U = Urasiel

ala = Alanien, arg = Arginien, asa = Asparaginsuur

asp = Asparagine, cys = Sisteïen, gom = Glutamiensuur

gl = Glutamine, gly = Glycine, his = Histidine

ile = Isoleucine, leu = Leucine, lys = Lisine, met = Metionien

phe = Fenielalanien, pro = Proline, ser = Serine, tyr = Tirosine

thr = Treonien, trp = Tryptofaan, val = Valine.

Dit verskaf verdere bewyse vir die drievoudige aard van die kodes. Eenvoudige trinukleotiede van bekende lineêre rangskikking is maklik om te sintetiseer. Die trinukleotiedgroep met 'n bekende basisvolgorde is eksperimenteel getoets vir hul spesifisiteit vir spesifieke tipe aminosuur. Deur volgorde-analise van so 'n stelsel kon die presiese kodes dus bepaal word.

Dit staan ​​bekend as bindtegniek. Eintlik het hierdie tegniek baie gehelp om die werklike kode uit te werk. Deur hierdie proses is al die 64 moontlike drieling afsonderlik getoets vir 20 aminosure en kondone vir al die aminosure is toegeken. Figuur 21.3 toon verskillende aminosure en hul onderskeie kodons.

In die Fig. 21.3 word drielinge UAG, UAA en UGA in mRNA genoem nie-sin kodons of kettingtermineerders (C.T.) aangesien hulle nie vir enige aminosuur kodeer nie en hul teenwoordigheid in mRNA lei tot die stop van polipeptiedvorming. Die drieling AUG en GUG blyk twee rolle te hê, eerstens, hulle tree op as ketting-inisieerders (C.I.) en tweedens, soos spesifiseer van aminosuur metionien en valien onderskeidelik.

Die genetiese kodes is universeel, dit wil sê die kodes wat spesifiek is vir spesifieke aminosure in bakterieë is ook spesifiek vir daardie einste aminosure in mens, muis, voëls en alle ander lewende organismes.

Dr. Khorana het ook onafhanklik aan die genetiese kodes gewerk. Hy was beïndruk deur Dr Nirenberg’s tegniek, maar hy het sy eie metode ontwikkel om die kode te kraak. Hy het DNA van bekende volgorde gesintetiseer en dit by die selvrye sisteem gevoeg om boodskapper-RNA van bekende volgorde te kry.

Hy het begin deur net twee DNS-basisse aan mekaar te koppel en dan die dubbelplate te polimeer om 'n lang DNS-string te gee wat 'n alternatiewe volgorde van die twee basisse bevat (sê byvoorbeeld AC—AC—AC—AC—AC—AC).

Deur trinukleotiedevolgordes soos TTC/AAG, en selfs tetranukleotiedvolgordes soos TTAC/AATG te herhaal, het hy in laboratorium agt soorte kunsmatige DNA gesintetiseer met bekende basisvolgordes wat soos volg is:

Die sintetiese mRNA gesintetiseer op die oppervlak van sintetiese DNA met 'n bekende basisvolgorde het dan die proteïensintese gerig. Uit die volgorde van aminosure in die proteïenmolekules wat so verkry is, kon die spesifieke kodes direk daarvoor vasgestel word. Deur hierdie prosedure te gebruik, het Khorana al die 64 kodons gevestig. Sommige van die opvallende kenmerke van hierdie eksperiment word hier opgesom.

1. Deur twee nukleotiedvolgordes in RNA te herhaal, is polipeptiede met twee aminosure in afwisselende rangskikking verkry, bv.

2. Herhalende trinukleotiedvolgordes het 'n mengsel van drie proteïene opgelewer, elk met 'n aantal spesifieke aminosure, byvoorbeeld die herhalende volgorde—CUU het proteïen met polifenielalanien opgelewer, proteïen met polileusien en proteïen met serien.

3. Herhalende tetranukleotiedvolgordes het hy proteïene gekry met herhaalde 4 aminosure volgorde soos byvoorbeeld:

Hierdie eksperimente het sonder twyfel 64 verskillende kodes vasgestel. Dit word in Fig. 21.3 getoon.

In 1959, toe Nirenberg daaraan gedink het om RNA van bekende volgorde te gebruik om kodewoorde uit te werk. Dr. Robert Holley en sy span by Cornell Universiteit het die uiters dapper besluit geneem om die struktuur van natuurlik voorkomende RNA uit te werk. Hulle het die kleinste vorm van RNA (tRNA) gekies en het van plan om dit van gis te isoleer. Teen 1962 het hulle 3 gis-tRNA's geïsoleer en gesuiwer.

Die gesuiwerde tRNA's is toe deur spesiale tegnieke in segmente verdeel en dan het hulle stuk vir stuk begin uitwerk, die volgorde van basisse langs die ruggraat van een van hulle. In die basisvolgorde van tRNA, wat die aminosuur alanien dra, was daar 'n streek wat die drieling CGI (I=Inosien 'n chemiese verbinding soortgelyk aan G) of feitlik GGG bevat het.

Daardie drieling het opgetree as adapter-nukleotied-triplet of antikodon vir alanien. Teen Maart 1965 het hulle in hul pogings geslaag en antikodons vir alle aminosure is geïdentifiseer. Deur die struktuur van rRNA-molekule te bepaal, het Dr. Holley en sy kollegas getoon dat dit moontlik was om met dieselfde tegniek ook ander nukleïensure te ontleed.

Vir hul briljante analitiese werk is dr. Holley, dr. Hargovind Khorana en dr. Nirenberg aangewys as medewenners van die 1968 Nobelprys.


Primêre struktuur bestaan ​​uit 'n lineêre volgorde van nukleotiede wat deur fosfodiesterbinding aan mekaar gekoppel is. Dit is hierdie lineêre volgorde van nukleotiede wat die primêre struktuur van DNA of RNA uitmaak. Nukleotiede bestaan ​​uit 3 komponente:

Die stikstofbasisse adenien en guanien is purien in struktuur en vorm 'n glikosidiese binding tussen hul 9 stikstof en die 1' -OH-groep van die deoksiribose. Sitosien, timien en uracil is pirimidiene, vandaar die glikosidiese bindings vorm tussen hul 1 stikstof en die 1' -OH van die deoksiribose. Vir beide die purien- en pirimidienbasisse vorm die fosfaatgroep 'n binding met die deoksiribosesuiker deur 'n esterbinding tussen een van sy negatief gelaaide suurstofgroepe en die 5' -OH van die suiker. [2] Die polariteit in DNS en RNA word afgelei van die suurstof- en stikstofatome in die ruggraat. Nukleïensure word gevorm wanneer nukleotiede bymekaar kom deur fosfodiester-bindings tussen die 5'- en 3'-koolstofatome. [3] 'n Nukleïensuurvolgorde is die volgorde van nukleotiede binne 'n DNA (GACT) of RNA (GACU) molekule wat deur 'n reeks letters bepaal word. Sekwensies word vanaf die 5'- tot 3'-kant aangebied en bepaal die kovalente struktuur van die hele molekule. Rye kan aanvullend tot 'n ander volgorde wees deurdat die basis op elke posisie komplementêr is sowel as in die omgekeerde volgorde. 'n Voorbeeld van 'n komplementêre volgorde tot AGCT is TCGA. DNA is dubbelstrengs en bevat beide 'n sintuigstring en 'n antisense string. Daarom sal die komplementêre volgorde tot die sintuigstring wees. [4]

Komplekse met alkalimetaalione Edit

Daar is drie potensiële metaalbindende groepe op nukleïensure: fosfaat-, suiker- en basisdele. Vastetoestandstruktuur van komplekse met alkalimetaalione is hersien. [6]

DNA wysig

Sekondêre struktuur is die stel interaksies tussen basisse, dit wil sê watter dele van stringe aan mekaar gebind is. In DNA-dubbelheliks word die twee DNA-stringe deur waterstofbindings bymekaar gehou. Die nukleotiede op een string basispaar met die nukleotied op die ander string. Die sekondêre struktuur is verantwoordelik vir die vorm wat die nukleïensuur aanneem. Die basisse in die DNA word geklassifiseer as puriene en pirimidiene. Die puriene is adenien en guanien. Puriene bestaan ​​uit 'n dubbelringstruktuur, 'n sesledige en 'n vyfledige ring wat stikstof bevat. Die pirimidiene is sitosien en timien. Dit het 'n enkelringstruktuur, 'n sesledige ring wat stikstof bevat. ’n Purienbasis pare altyd met ’n pirimidienbasis (guanien (G) pare met sitosien (C) en adenien (A) pare met timien (T) of uracil (U)). DNS se sekondêre struktuur word hoofsaaklik bepaal deur basisparing van die twee polinukleotiedstringe wat om mekaar gedraai is om 'n dubbelheliks te vorm. Alhoewel die twee stringe in lyn gebring word deur waterstofbindings in basispare, is die sterker kragte wat die twee stringe bymekaar hou, die stapelinteraksies tussen die basisse. Hierdie stapel-interaksies word gestabiliseer deur Van der Waals-kragte en hidrofobiese interaksies, en toon 'n groot hoeveelheid plaaslike strukturele veranderlikheid. [7] Daar is ook twee groewe in die dubbelheliks, wat op grond van hul relatiewe grootte hoofgroef en klein groef genoem word.

RNA wysig

Die sekondêre struktuur van RNA bestaan ​​uit 'n enkele polinukleotied. Basisparing in RNA vind plaas wanneer RNA tussen komplementariteitstreke vou. Beide enkel- en dubbelstrengige streke word dikwels in RNA-molekules aangetref.

Die vier basiese elemente in die sekondêre struktuur van RNA is:

Die antiparallelle stringe vorm 'n heliese vorm. [3] Bulte en interne lusse word gevorm deur skeiding van die dubbelheliese kanaal op óf een string (bult) óf op albei stringe (interne lusse) deur ongepaarde nukleotiede.

Stam-lus of haarnaald lus is die mees algemene element van RNA sekondêre struktuur. [8] Stam-lus word gevorm wanneer die RNA-kettings op hulself terugvou om 'n dubbelheliese kanaal te vorm wat die 'stam' genoem word, die ongepaarde nukleotiede vorm enkelstrengige streek wat die 'lus' genoem word. [9] 'n Tetraloop is 'n vier-basispare haarnaald-RNA-struktuur. Daar is drie algemene families van tetraloop in ribosomale RNA: UNCG, GNRA, en CUUG (N is een van die vier nukleotiede en R is 'n purien). UNCG is die mees stabiele tetraloop. [10]

Pseudoknot is 'n RNA sekondêre struktuur wat die eerste keer in raapgeel mosaïekvirus geïdentifiseer is. [11] Pseudoknote word gevorm wanneer nukleotiede van die haarnaald-lus-paar met 'n enkelstrengige streek buite die haarnaald om 'n heliese segment te vorm. H-tipe vou pseudoknote word die beste gekenmerk. In H-tipe vou, nukleotiede in die haarnaald-lus-paar met die basisse buite die haarnaaldstam wat tweede steel en lus vorm. Dit veroorsaak die vorming van pseudoknote met twee stamme en twee lusse. [12] Skuilknope is funksionele elemente in RNA-struktuur met uiteenlopende funksie en word in die meeste klasse RNA aangetref.

Sekondêre struktuur van RNA kan voorspel word deur eksperimentele data oor die sekondêre struktuurelemente, helikse, lusse en bulte. DotKnot-PW metode word gebruik vir vergelykende pseudoknots voorspelling. Die hoofpunte in die DotKnot-PW-metode is om die ooreenkomste wat in stamme, sekondêre elemente en H-tipe pseudoknote gevind word, aan te teken. [13]

Tersiêre struktuur verwys na die liggings van die atome in driedimensionele ruimte, met inagneming van geometriese en steriese beperkings. Dit is 'n hoër orde as die sekondêre struktuur, waarin grootskaalse vou in 'n lineêre polimeer plaasvind en die hele ketting in 'n spesifieke 3-dimensionele vorm gevou word. Daar is 4 areas waarin die strukturele vorme van DNA kan verskil.

  1. Handigheid – regs of links
  2. Lengte van die heliksdraai
  3. Aantal basispare per beurt
  4. Verskil in grootte tussen die groot en klein groewe [3]

Die tersiêre rangskikking van DNA se dubbelheliks in die ruimte sluit B-DNA, A-DNA en Z-DNA in.

B-DNA is die mees algemene vorm van DNA in vivo en is 'n nouer, verlengde heliks as A-DNA. Sy wye groot groef maak dit meer toeganklik vir proteïene. Aan die ander kant het dit 'n smal klein groef. B-DNA se bevoordeelde konformasies vind plaas by hoë waterkonsentrasies, die hidrasie van die klein groef blyk B-DNS te bevoordeel. B-DNA basispare is byna loodreg op die heliks-as. Die suikerrimpel wat die vorm van die a-heliks bepaal, of die heliks in die A-vorm of in die B-vorm sal bestaan, kom by die C2'-endo voor. [14]

A-DNA, is 'n vorm van die DNA-dupleks wat onder dehidrerende toestande waargeneem word. Dit is korter en breër as B-DNA. RNA neem hierdie dubbelheliese vorm aan, en RNA-DNS-duplekse is meestal A-vorm, maar B-vorm RNA-DNS-duplekse is waargeneem. [15] In gelokaliseerde enkelstring dinukleotiedkontekste kan RNA ook die B-vorm aanneem sonder om met DNS te koppel. [16] A-DNS het 'n diep, nou hoofgroef wat dit nie maklik vir proteïene toeganklik maak nie. Aan die ander kant maak sy wye, vlak klein groef dit toeganklik vir proteïene, maar met laer inligting-inhoud as die hoofgroef. Sy gunsteling bouvorm is by lae waterkonsentrasies. A-DNS's basispare is gekantel relatief tot die heliks-as, en word van die as verplaas. Die suikerpucker vind plaas by die C3'-endo en in RNA inhibeer 2'-OH C2'-endo-konformasie. [14] A-DNS wat lank as weinig meer as 'n laboratoriumkunsstuk beskou is, is nou bekend dat dit verskeie biologiese funksies het.

Z-DNA is 'n relatief skaars linkshandige dubbelheliks. Gegewe die regte volgorde en superheliese spanning, kan dit in vivo gevorm word, maar die funksie daarvan is onduidelik. Dit het 'n nouer, meer langwerpige heliks as A of B. Z-DNA se hoofgroef is nie regtig 'n groef nie, en dit het 'n smal klein groef. Die mees bevoordeelde konformasie vind plaas wanneer daar hoë soutkonsentrasies is. Daar is 'n paar basisvervangings, maar hulle benodig 'n afwisselende purien-pirimidienvolgorde. Die N2-amino van G H-bindings aan 5' PO, wat die stadige uitruiling van protone en die behoefte aan die G purien verduidelik. Z-DNA basispare is byna loodreg op die heliks-as. Z-DNA bevat nie enkele basispare nie, maar eerder 'n GpC-herhaling met P-P-afstande wat verskil vir GpC en CpG. Op die GpC-stapel is daar goeie basis-oorvleueling, terwyl daar op die CpG-stapel minder oorvleueling is. Z-DNA se sigsag-ruggraat is te wyte aan die C-suiker-konformasie wat kompenseer vir G-glikosidiese binding-konformasie. Die konformasie van G is syn, C2'-endo vir C is dit anti, C3'-endo. [14]

’n Lineêre DNS-molekule met vrye punte kan roteer, om aan te pas by veranderinge van verskeie dinamiese prosesse in die sel, deur te verander hoeveel keer die twee kettings van sy dubbelheliks om mekaar draai. Sommige DNA-molekules is sirkelvormig en is topologies beperk. Meer onlangs is sirkelvormige RNA ook beskryf as 'n natuurlike deurdringende klas van nukleïensure, wat in baie organismes uitgedruk word (sien CircRNA).

'n Kovalent geslote, sirkelvormige DNS (ook bekend as cccDNA) word topologies beperk aangesien die aantal kere wat die kettings om mekaar gedraai is nie kan verander nie. Hierdie cccDNA kan supercoil wees, wat die tersiêre struktuur van DNA is. Supercoiling word gekenmerk deur die skakelgetal, draai en wriemel. Die skakelgetal (Lk) vir sirkelvormige DNA word gedefinieer as die aantal kere wat een string deur die ander string sal moet gaan om die twee stringe heeltemal te skei. Die skakelgetal vir sirkelvormige DNA kan slegs verander word deur 'n kovalente binding in een van die twee stringe te breek. Altyd 'n heelgetal, die skakelgetal van 'n cccDNA is die som van twee komponente: kronkels (Tw) en writhes (Wr). [17]

Draaiings is die aantal kere wat die twee stringe DNA om mekaar gedraai word. Writhes is die aantal kere wat die DNA-heliks oor homself kruis. DNS in selle is negatief opgerol en het die neiging om te ontspan. Gevolglik is die skeiding van stringe makliker in negatief supergedraaide DNA as in ontspanne DNA. Die twee komponente van supergedraaide DNA is solenoïed en plektonemies. Die pektonemiese superspoel word in prokariote aangetref, terwyl die solenoïdale superspoeling meestal in eukariote gesien word.

Die kwaternêre struktuur van nukleïensure is soortgelyk aan dié van proteïen kwaternêre struktuur. Alhoewel sommige van die konsepte nie presies dieselfde is nie, verwys die kwaternêre struktuur na 'n hoër vlak van organisasie van nukleïensure. Boonop verwys dit na interaksies van die nukleïensure met ander molekules. Die mees algemeen gesiene vorm van hoër-vlak organisasie van nukleïensure word gesien in die vorm van chromatien wat lei tot sy interaksies met die klein proteïene histone. Die kwaternêre struktuur verwys ook na die interaksies tussen afsonderlike RNA-eenhede in die ribosoom of spliceosoom. [18]


Die genetiese kode in DNA

Die instruksies vir die konstruksie van proteïene word in DNS geskryf deur die genetiese kode te gebruik. Meer spesifiek bevat die volgorde van basisse wat aan die suikerfosfaatruggraat van die dubbelheliks gebind is inligting in die vorm van driebasiskodons wat die volgorde spesifiseer van aminosure wat in die konstruksie van proteïene gebruik moet word.

Die volgorde van basisse in DNS werk as 'n ware kode deurdat dit die inligting bevat wat nodig is om 'n proteïen te bou wat uitgedruk word in 'n vierletter-alfabet van basisse wat na mRNA getranskribeer word en dan vertaal word na die twintig-aminosuur-alfabet wat nodig is om te bou die proteïen. Om te sê dat dit 'n ware kode is, behels die idee dat die kode vry en onbeperk is, kan enige van die vier basisse in enige van die posisies in die volgorde van basisse geplaas word. Hulle volgorde word nie deur die chemiese binding bepaal nie. There are hydrogen bonds between the base pairs and each base is bonded to the sugar phosphate backbone, but there are no bonds along the longitudional axis of DNA. The bases occur in the complementary base pairs A-T and G-C, but along the sequence on one side the bases can occur in any order, like the letters of a language used to compose words and sentences.


The Chemical Structure of DNA

Click to enlarge

Today’s post crosses over into the realm of biochemistry, with a look at the chemical structure of DNA, and its role in creating proteins in our cells. Of course, it’s not just in humans that DNA is found – it’s present in the cells of every multicellular life form on Earth. This graphic provides an overview of its common structure across these life forms, and a brief explanation of how it allows proteins to be generated.

DNA is found in the nucleus of cells in multicellular organisms, and was first isolated in 1869, by the Swiss physician Friedrich Miescher. However, its structure was not elucidated until almost a century later, in 1953. The authors of the paper in which this structure was suggested, James Watson & Francis Crick, are now household names, and won a Nobel prize for their work. This work, however, was heavily reliant on the work of another scientist, Rosalind Franklin.

Franklin herself was also investigating the structure of DNA, and it was her X-ray photograph, clearly showing the double helix structure of DNA, that greatly aided their work. She had yet to publish her findings when Watson and Crick obtained access to them, without her knowledge. However, her failure to win a Nobel prize is not an oversight, but merely a consequence of the committee’s policy that Nobel prizes cannot be awarded posthumously.

The double helix model of DNA (deoxyribonucleic acid) consists of two intertwined strands. These strands are made up of nucleotides, which themselves consist of three component parts: a sugar group, a phosphate group, and a base. The sugar and phosphate groups combined form the repeating ‘backbone’ of the DNA strands. There are four different bases that can potentially be attached to the sugar group: adenine, thymine, guanine and cytosine, given the designations A, T, G and C.

The bases are what allows the two strands of DNA to hold together. Strong intermolecular forces called hydrogen bonds between the bases on adjacent strands are responsible for this because of the structures of the different bases, adenine (A) always forms hydrogen bonds with thymine (T), whilst guanine (G) always forms hydrogen bonds with cytosine (C). In human DNA, on average there are 150 million base pairs in a single molecule – so many more than shown here!

The cells in your body constantly divide, regenerate, and die, but for this process to occur, the DNA within the cell must be able to replicate itself. During cell division, the two strands of DNA split, and the two single strands can then be used as a template in order to construct a new version of the complimentary strand. As A always pairs with T, and G always pairs with C, it’s possible to work out the sequence of bases on the one strand using the opposite strand, and it’s this that allows the DNA to replicate itself. This process is carried out by a family of enzymes called DNA polymerases.

When DNA is used to create proteins, the two strands must also split. In this case, however, the DNA’s code is copied to mRNA (messenger ribonucleic acid), a process known as ‘transcription’. RNA’s structure is very similar to that of DNA, but with a few key differences. Firstly, it contains a different sugar group in the sugar phosphate backbone of the molecule: ribose instead of deoxyribose. Secondly, it still uses the bases A, G and C, but instead of the base T, it uses uracil, U. The structure of uracil is very similar to thymine, with the absence of a methyl (CH3) group being the only difference.

Once the DNA’s nucleotides have been copied, the mRNA can leave the nucleus of the cell, and makes its way to the cytoplasm, where protein synthesis takes place. Here, complicated molecules called ribosomes ‘read’ the sequence of bases on the mRNA molecule. Individual amino acids, which combined make up proteins, are coded for by three letter sections of the mRNA strand. The different possible codes, and the amino acids they code for, were summarised in a previous post that looked at amino acid structures. A different type of RNA, transfer RNA, is responsible for transporting amino acids to the mRNA, and allowing them to join together.

This process isn’t always flawless, however. Errors can occur in copying DNA’s sequence to mRNA, and these random errors are referred to as mutations. The errors can be in the form of a changed base, or even a deleted or added base. Some chemicals, and radiation, can induce these changes, but they can also happen in the absence of these external effects. They can lead to an amino acid’s code being changed to that of another, or even rendered unreadable. A number of diseases can result from mutations during DNA replication, including cystic fibrosis, and sickle-cell anaemia, but it’s worth noting that mutations can also have positive effects.

Though there are only 20 amino acids, the human body can combine them to produce a staggering figure of approximately 100,000 proteins. Their creation is a continuous process, and a single protein chain can have 10-15 amino acids added to it per second via the process outline above. As the purpose of this post was primarily to examine the chemical structure of DNA, the discussion of replication and protein synthesis has been kept brief and relatively simplistic. If you’re interested in reading more into the subject, check out the links provided below!

Thanks goes to Liam Thompson for the help with the research for this post, and providing an incredibly useful simple overview of the process of protein synthesis from DNA.


The Bases A, C, G, and T

Two ring structures are found in the bases. C (cytosine) and T (thymine) have a single six membered ring, called a pyrimidine ring. A (adenine) and G (guanidine) have two rings joined together. This unit is called a purine ring. C and T are called pyrimidine bases A and G are called purine bases. Here are the structures:

In each of these bases there is a secondary amine whose nitrogen forms a bond to the anomeric carbon of a deoxyribose in the DNA backbone. We can relate the chemistry of the formation of this linkage to the formation of a glycoside (acetal) from glucose (hemiacetal) and an alcohol. The difference is that in the current case the nucleophile is the secondary amine nitrogen of a base rather than the oxygen of an alcohol. An example of four bases attached in this way is:

The "word" here is CACT. Recall that the DNA backbone is very long, and it is clear that even with only a four letter alphabet, a great deal of information can be carried by DNA


Puriene

Purines are heterocyclic aromatic compounds with an imidazole ring fused to the pyrimidine ring. They were first synthesized by Emil Fischer in 1899, by treating uric acid with phosphorous pentachloride to produce purines. They are also naturally found in high concentrations in meat and meat products. There are two types of purines in the form of DNA bases.

Adenien

Its chemical IUPAC name is 9H-purin-6-amine. It is a purine derivative with an additional amine group at the 6th position. It was named and identified by Albrecht Kossel in 1885. He isolated it from pancreatic tissues. It forms the nucleotide, adenine. Its triphosphate form, adenosine triphosphate (ATP) is extensively utilized in cellular processes as the basic form of chemical energy. In its other phosphate forms, it plays the role of catalyst and co-factor. It occurs in DNA as deoxyadenosine triphosphate (dATP).

Guanine

Its chemical IUPAC name is 2-amino-1H-purin-6(9H)-one. It is a purine derivative with a carbonyl bond at the 6th position. It was first isolated from excreta of sea birds in 1844, and termed as guano. Later Emil Fischer determined its structure, and synthesized it from uric acid. It forms the nucleotide, guanine. Its monophosphate form, guanosine monophosphate (GMP), when salted out, acts as a flavoring agent that imparts an umami taste. It occurs in DNA as deoxyguanosine triphosphate.


Part III: Frontiers &mdash

Eukaryotic chromosomes are long, linear DNA molecules bacterial chromosomes are frequently circles of DNA. However, scientists realized that the simple rules of DNA base pairing could be exploited to create much more complex shapes than are found in nature. Ned Seeman, in 1982, first began exploring the use of DNA as a building material for applications in nanotechnology. Today, DNA nanotechnology has become a booming area of research, attracting both engineers and biologists.

The basic principle of DNA nanotechnology is based upon creating branch points that connect multiple strands of DNA. Biological DNA consists of two strands that are fully complementary, creating a linear molecule. However, four strands of DNA that are partially complementary can interact in the manner shown in Figure 34 . Two of the DNA strands act as "staples" that bridge two non-complementary DNA strands that normally would not interact. With this simple design, a simple 2D shape is created: a rectangle of two tightly packed helices. Staples also can connect more than two segments of DNA and thus create more complicated 3D shapes.

Figure 34 This illustration shows how DNA staples can bridge two non-complementary DNA strands and create two side-by-side helices.

DNA Origami

A popular method of fabricating 3D DNA nanostructures is called DNA origami , which was invented by Paul Rothemund in 2006. To showcase this nanoscale design, Rothmund made smiley faces out of DNA, which were featured on the cover of Nature, the same journal that published the trio of pivotal DNA structure papers from Watson/Crick, Franklin, and Wilkins in 1953 ( Figure 35 ). Keep in mind that these DNA smiley faces are only ∼100 nm in diameter, which is much smaller than a typical human cell (30,000 nm in diameter).

Figure 35 The cover of Nature from March 16, 2006, featuring the work of Paul Rothemund entitled "Folding DNA to create nanoscale shapes and patterns." See reference list. Permission obtained from Rothemund and Nature.

DNA origami involves mixing two components: (1) a very long, single-stranded "scaffold" strand that on its own just forms a very floppy circle, and (2) a mixture of short oligonucleotides of single-stranded DNA (staples) that are complementary to different regions of the scaffold strand. When the staples and scaffold form helices, different parts of the circle are brought together, folding it into a very specific shape (hence the whimsical name "origami"). One advantage of the origami approach is that different shapes can be created from one scaffold with different oligonucleotide staples ( Figure 36 ).

Figure 36 DNA origami involves combining one large single-stranded DNA "scaffold" (7000 bases) with many short single-stranded DNA "staples." By changing the staples, one scaffold can fold into multiple shapes.

The actual strand-routing diagram of a DNA origami design might look something like that shown in Figure 37 :

Figure 37 Diagram of a DNA origami design the staples connect to two or three different regions of the scaffold creating helices that pack into a specific shape. Artistic redrawing from a figure from the 2006 Rothemund paper (see reference list).

So how did Rothemund make the DNA smiley face? The process involves two important steps.

The genome of a small single-stranded DNA virus (∼8000 nucleotides) is typically used as the scaffold. The virus can be prepared in large quantities and its DNA purified. The custom-designed DNA staples are made by humans, and not nature. Many commercial companies use automatic machines that execute a sequence of chemical reactions to synthesize single-stranded DNA with any custom sequence. Today, scientists design a specific DNA sequence on their computer (up to a few thousand nucleotides at a cost of ∼10 cents per base), send in the order electronically to the DNA synthesis company, and a tube of billions of identical single-stranded DNA molecules arrives by courier to their lab bench a few days later ( Figure 38 ). Making custom DNA sequences has certainly become very easy. On the other hand, designing the sequences of ∼200 DNA staples that will produce a specific shape is not trivial. However, computer software (cadnano https://cadnano.org/) is available to facilitate this process.

Figure 38 The workflow of designing and receiving DNA for research.

The single-stranded DNA scaffold and staples then need to be combined. The DNA mixture is first heated to break apart any preexisting base pairs, and then cooled down to room temperature to allow base pairing between the scaffold and staples, as depicted in the animation in Video 8 .

Video 8 Animation of the formation of DNA origami. The single-stranded scaffold and the staples strands come together through Watson–Crick base pairing to form helices that pack into a particular 2D or 3D shape. Animation by Shawn Douglas.

In the case of Rothemund’s smiley faces, ∼200 distinct DNA oligonucleotide staples were combined with one long (∼7000 nucleotides) single-stranded DNA scaffold. Helices in adjacent rows of the smiley face are connected through branch points created by the DNA staples ( Figure 39 ).

Figure 39 Figure 39: The making of a smiley face DNA origami through the combination of many short oligonucleotide staples with a long scaffold strand.

The last step is checking whether the DNA origami process actually worked. As the structures are small (one ten-millionth of a meter), one needs a high-resolution method like electron microscopy or atomic force microscopy to image these structures, as shown in Figure 40 .

Figure 40 An image of DNA origami smiley faces using atomic force microscopy. The scale bar is 100 nanometers. Image obtained from the 2006 Rothemund paper (see reference list) with permission.

A DNA Origami Nanorobot that Inhibits Cancer Cell Proliferation

DNA origami is being used in many creative ways. One example is a DNA origami nanorobot was designed to halt the division of cancer cells (see the Guided Paper by Shawn Douglas, Ido Bachelet, and George Church). The nanorobot carries a "payload" (an inhibitory antibody ), which it delivers if, and only if, it encounters a cell with a molecular signature characteristic of cancer.

The nanorobot consists of two halves that come together to form a barrel. The two halves are closed with two pairs of complementary DNA strands placed at the mouth of the barrel ( Figure 41 ). The middle of the barrel contains growth-inhibiting antibodies that are attached to the DNA. When the antibodies are trapped inside of the barrel, they cannot interact with cells as a result, the DNA nanorobot in the closed barrel form is harmless.

Figure 41 The nanorobot made by Shawn Douglas and colleagues combines a single ∼7000 nucleotide scaffold with ∼200 short oligonucleotide staples. It is shown here in its closed form. Antibody fragments (portions of a full antibody, called single-chain antibodies) that can inhibit the growth of cancer cells are present inside the barrel.

The trick, then, is to get the cancer cell to open up the nanorobot and expose the inhibitory antibodies. To accomplish this, Douglas developed a clever unlocking mechanism. One DNA strand of the pair that keeps the barrel closed has a special capability of binding to a specific protein expressed on a cancer cell. This type of DNA sequence is called an aptamer . When its protein partner is present, the aptamer will let go of its complementary DNA strand and wrap around the protein instead ( Figure 42 ).

Figure 42 A DNA aptamer is selected for an ability to bind to a specific protein as well as to a complementary DNA strand. Binding to the protein is tighter and thus will be preferred.

The DNA aptamer provides an "intelligent" lock that keeps the DNA nanorobot closed unless it encounters a protein from a cancer cell ( Figure 43 ). The protein on the cancer cell acts as a "key" that unlocks and opens up the two halves of the nanorobot, exposing the payload inside.

Figure 43 The nanorobot is normally closed via two pairs of complementary DNA strands at the mouth of the barrel. One strand of each pair is an aptamer that can bind to a target protein. When that protein is present, the aptamer will bind to it, unlocking the nanorobot.

The nanorobot also can be designed to be extra smart, and open up if, and only if, it detects two different proteins instead of one. In this case, two different DNA aptamers, which recognize different proteins, are deployed on either side of the barrel mouth. If one aptamer binds its partner protein but the second aptamer does not, then the barrel will remain closed since only one side is unlocked. Both proteins must be present to unlock and fully open the barrel. This dual recognition aptamer system offers improved specificity for a target. For example, a combination of two cell surface proteins better defines a unique molecular signature of a cancer cell than a single protein.

With these design principles in place, Douglas built a nanorobot that would battle leukemia cells ( white blood cell -derived cancer) ( Figure 44 ). The nanorobot’s payload was an antibody that inactivates a cell surface protein that promotes cell proliferation. The opening and closing of the nanorobot was controlled by two DNA aptamers that recognize two different cell surface proteins that are abundant on leukemia cells but not on normal white blood cells. Tests of the nanorobot (see the Guided Paper) confirmed that it opens efficiently in the presence of leukemic cells but not normal white blood cells.

Figure 44 The DNA nanorobot in action. Two different locking aptamers recognize different proteins on a cancer cell. In the presence of these proteins, the nanorobot opens up and exposes antibodies that bind to another target protein on the cancer cell, which results in a suppression of cell division.

Finally, the nanorobot was ready for testing with leukemia cells growing in cell culture. Three versions of the nanorobot were tested. The "gated" nanorobot had the dual aptamers designed to open up in response to encountering two specific proteins on the surface of the leukemic cells. Two other nanorobots were tested to assess how well the cell-specific locking mechanism worked. One was permanently locked using non-aptamer DNA strands. The second was permanently open as it lacked locking strands. The results are shown in Figure 45 . The "always locked" nanorobots had no or little effect on cell division at any DNA nanorobot concentration. In contrast, the "always unlocked" nanorobot inhibited cell division with increasing concentrations of the nanorobot. The "gated" nanorobot also inhibited cell division, indicating that it must have opened up when it encountered the cancer cells. While the DNA nanorobot has not yet been used to treat cancer patients (only cancer cells growing on a dish in the lab), it provides an example of how "smart" nanoscale devices can be created with DNA origami.

Figure 45 The effects of different nanorobots on cancer cell proliferation (cell division). The "gated" nanorobot is the one shown in Figure 44. The "always locked" nanorobot has locking DNA strands that are not aptamers. The "always unlocked" nanorobot does not have locking DNA strands.

Explorer’s Question: Why do you think that the growth inhibition curve for the "gated" nanorobot is shifted to the right of the "unlocked" nanorobot?

Antwoord: The rightward shift of the curve indicates that the gated nanorobot is less efficient in inhibiting cell division for example, at 1 nM concentration, the "gated" nanorobot was ineffective while the "always unlocked" nanorobot had an effect. This result indicates that the gate might be a bit "sticky" and that not all nanorobots open up when they encounter the cancer cells.

Explorer’s Question: If the "gated" nanorobot is less efficient, why not just use the "unlocked" nanorobot for inhibiting cancer cells?

Antwoord: There is a trade-off between efficiency and specificity. It is desirable to kill the leukemic cells, but undesirable to react with normal white blood cells or bone marrow cells that make white blood cells. The "always unlocked" nanorobots, while more efficient, have the potential to affect normal cells. The "gated" nanorobots provide specificity by targeting the inhibitory payload antibodies to the cancer cells.

Explorer’s Question: Why are only 50% of the cancer cells inhibited in their growth?

Antwoord: The graph suggests the effect of the nanorobots reaches a plateau at ∼50% growth inhibition (although it would be useful to have additional data points to establish this conclusion in a more convincing manner). There are several possibilities. First, it is possible that the exposure time (3 days) of the nanorobots was too short and that greater inhibition could be achieved with a longer incubation time. Second, the nanorobot inhibition may be partial 50% of the cancer cells escape nanorobot inhibition and divide. Third, a subset of cancer cells may be resistant to the nanorobot (resistance is a common property of cancer cells see Narrative on Cancer by Mike Bishop ). This third possibility could be confirmed by exposing cancer cells to nanorobots and selecting the survivors. If the survivors are re-exposed to nanorobots and are unaffected, then they are a resistant subpopulation. As an example of resistance, cells might have stopped producing one the protein "keys" needed to unlock the nanorobot.

Explorer’s Question: What other controls could you imagine performing to better understand the specificity of the nanorobots?

Antwoord: In their paper, the authors showed that the "gated" nanorobot did not bind to normal white blood cells, but it might be useful to test for functional effects as well. For example, T cells (a type of white blood cell) can be induced to divide in response to an invading organism. Does the nanorobot affect the division of T cells? One also might want to know if the "gated" nanorobot opened in response to the correct aptamer-targeting proteins on the leukemia cells. Using current CRISPR gene editing technologies (see Narrative by Barrangou ), one can disrupt the genes for these two proteins in these cancer cells. In these gene-edited cells, the "gated nanorobot" should not be able to open and should not inhibit cell growth.

Undergraduates and DNA Nanotechnology

Does DNA origami and nanoscale biomolecular engineering sound cool? But alas only accessible to PhD scientists? Think again. Now undergraduates are getting into the game. Every year, undergraduates from around the world are designing amazing biomolecule-based devices, which include nanoscale robots, computers, and therapeutics, in a competition called BIOMOD. The selected teams of undergraduates then meet in person in San Francisco to share their ideas and potentially win a prize. You can learn about some of the amazing DNA origami ideas that undergraduate students have thought of in Video 9 .

Video 9 Undergraduate students share their ideas of DNA origami-based nanomachines in an international competition called BIOMOD.


DNA: ‘The Power of the Beautiful Experiment’

Matthew Cobb tells this story in his latest book, Life&rsquos Greatest Secret. Cobb, a professor of zoology at the University of Manchester, is a working geneticist. He is also a student of the history of science who has written several previous books on the history of biology. Life&rsquos Greatest Secret is aimed at the general reader who may have only a passing familiarity with biology, much less with the detailed molecular mechanics of how DNA does what it does. The book serves as a useful primer for those interested in the brave new world of genetic intervention made possible by the rise of biotechnology. But Cobb&rsquos book will also be of interest to professional scientists as it recounts events in one of the most transformative periods in the history of science: the rise of a molecular understanding of life.

Despite its dense historical detail, Life&rsquos Greatest Secret is an absorbing and, in places, thrilling book. The race to crack the genetic code is a story with considerable drama and it unfolds remarkably lucidly in Cobb&rsquos telling.

The rise of this style of thinking had everything to do with what was happening in other fields of science during and immediately after World War II. This period saw the emergence of two new sciences that focused on information. Claude Shannon and others articulated information theory, which quantified the amount of information that flows from sender to receiver during, say, electronic communication. And Norbert Weiner elaborated cybernetics, which formalized ideas like feedback loops, especially negative feedback loops. (A thermostat involves a feedback loop: a setting on a thermostat affects a room&rsquos temperature and the temperature then affects the thermostat.)

Following these developments, some scientists grew excited at the prospect that the mathematical abstractions developed in these fields might provide a radical new way to think about life. Organisms might not look like the stuff of equations that describe the flow of information but the new sciences hinted that they might well be. Information thinking, Cobb claims, played a crucial part in helping to define what came to be called the &ldquocoding problem,&rdquo a problem that dominated biology in the 1950s and 1960s.

So, finally, what are proteins? Proteins are long molecular chains made of many individual links called amino acids. Organisms use twenty different kinds of amino acid to build their proteins. Your beta-globin is a sequence of 146 amino acids in a particular order. If you were to switch any one amino acid in a protein with another amino acid, things might go terribly wrong. As Cobb notes, the normal and sickle-cell forms of beta-globin&mdashthe latter causes sickle-cell anemia&mdashdiffer by only a single amino acid.

Crick&rsquos letter was auctioned in 2013 for $6 million.

Several weeks after Watson and Crick published their double-helix paper, they wrote, &ldquoThe precise sequence of the bases is the code which carries the genetical information.&rdquo By 1957, Crick was emphasizing that by &ldquoinformation&rdquo he meant &ldquothe specification of the amino acid sequence of the protein.&rdquo These statements, Cobb says, are among some of the earliest explicit usages of the language of information by biologists. (The physicist Erwin Schrödinger had used somewhat similar language earlier.)

More important, the race to crack the code was on.

As biologists were soon to realize, that the coding problem could be stated simply did not mean that it could be solved easily. Much of Cobb&rsquos book is given over to the two main approaches that were taken: theory and experiment.

Fortunately, speculation about the code didn&rsquot occur in an empirical vacuum. Some possible coding schemes, for example, placed constraints on which amino acids could occur next to each other in a protein. But when biochemists characterized many proteins from many species they found that a given amino acid might be followed by any of the twenty amino acids.

The confusion would soon disappear. But its resolution would not involve the clever calculations of the theorists. Nor would it involve the usual suspects, a small circle of brilliant biologists that orbited the yet-more-brilliant Crick. Instead, the code would be cracked by an obscure team: Marshall Nirenberg and Heinrich Matthaei of the National Institutes of Health in Bethesda, Maryland. Nirenberg, the older of the pair, was so unknown that his application to a conference on the genetic code was rejected in 1961. As Cobb puts it, &ldquoIronically, while the great and the good of molecular biology were talking about the genetic code, Nirenberg and Matthaei were cracking it.&rdquo

Cold Spring Harbor Laboratory Press

&lsquoAn outline of how the genetic code works during protein synthesis&rsquo from Matthew Cobb&rsquos book Life&rsquos Greatest Secret. According to Cobb, a codon is &lsquoa sequence of three bases in a DNA or RNA molecule that codes for an amino acid,&rsquo and a ribosome is a &lsquocomplex RNA structure found in all cells that is the primary site of protein synthesis.&rsquo A polypeptide is a chain of amino acids.

The last third of Life&rsquos Greatest Secret is devoted to bringing the history of the molecular side of genetics up to date. Of the many discoveries that followed the cracking of the genetic code, perhaps the most fundamental was the finding that the code is nearly universal across all life on earth. (Some minor variants on the code exist.) This finding is of deep evolutionary significance. All of us&mdashbacteria, fungi, plants, and people&mdashshare the same code because we all share an ancestor that lived billions of years ago and that employed this code.

It&rsquos also reasonably clear why the code has remained fixed through this vast stretch of evolutionary time. If it were to change, with, say, GCA encoding something besides the usual amino acid alanine, the structure of hundreds or thousands of proteins would suddenly and simultaneously change, a certain formula for disaster for any organism that tried it. While there&rsquos no obvious physical or chemical reason why certain letters of DNA encode certain amino acids, once life settled on a code early in evolutionary history, it couldn&rsquot be changed without catastrophic consequences. Crick called this the &ldquofrozen accident&rdquo hypothesis.

In the last part of Life&rsquos Greatest Secret Cobb turns to developments of social significance that followed from the cracking of the code particularly and advances in molecular genetics generally. Two are of special importance: the creation of genetically modified (GM ) crops and the attempt to cure human genetic disease.

By contrast, physicians have had mixed success in using genetic technologies to cure inherited diseases in human beings. (These interventions are usually intended to inject a normal, healthy copy of a gene into a patient&rsquos diseased tissue, such as the liver, not into the patient&rsquos eggs or sperm. As Cobb notes, the genetic modification will not, therefore, affect future generations.) Genetic technologies have not, so far, transformed medicine in the same way that they have agriculture.

While these more modern developments in molecular genetics are certainly important, the last third of Cobb&rsquos book can&rsquot compete with the rest. These later chapters are in places a bit rambling and sometimes read more like a textbook and less like the historical thriller of earlier chapters. Life&rsquos Greatest Secret might have been better off without this material. But it would be churlish to make too much of that. On the whole Cobb tells his story beautifully and his book is a pleasure to read. Packed with fascinating detail, Life&rsquos Greatest Secret is a major accomplishment, particularly for an author who is also a practicing scientist. Though I like to think that I have a good grasp of the history of genetics and evolutionary biology, I was repeatedly surprised by events in Cobb&rsquos tale.

Several major themes emerge from Life&rsquos Greatest Secret. The first concerns the influence of information theory and cybernetic thinking in biology. These disciplines, Cobb concludes, had an important part in twentieth-century biology &ldquobut not in the way in which their partisans might have hoped for.&rdquo In the end, the information sciences provided biologists with loose but useful metaphors and analogies, a language that allowed scientists to think and speak in new ways. But the high-powered mathematics of these fields proved mostly impotent in biology. No one, for instance, used Shannon&rsquos equations to say anything especially interesting about organisms. (A fact that didn&rsquot surprise Shannon himself, who was skeptical all along of this attempted use of his theory.) The history of science, like the history of anything, is characterized by considerable nuance, and one subtlety is that the information sciences were, in one sense, critical to the rise of modern biology and, in another sense, beside the point.

A second theme concerns the respective roles of theory (of any sort) versus experiment in biology. In the early 1960s, mathematicians confidently declared that &ldquoit will be interesting to see how much of the final solution [to the coding problem] will be proposed by mathematicians before the experimentalists find it.&rdquo As Cobb concludes, the &ldquoanswer&hellipwas simple: not one single part of it.&rdquo

The interesting question is why theory failed here. Part of the answer, as Cobb emphasizes, is related to Crick&rsquos idea of the frozen accident. The genetic code seems at least partly arbitrary. It represents a half-decent arrangement arrived at by the imperfect, tinkering process of evolution by natural selection and, once settled on, it couldn&rsquot be &ldquoimproved,&rdquo or made somehow more systematic. In such a situation theory is likely useless.

I suspect there&rsquos another, related, reason that theory contributed so little to cracking the code. There was, at bottom, a mismatch between the nature of the problem and the nature of much biological theory. Successful theory in biology typically plays a different part than does successful theory in, say, physics. Theory in biology often guides thought, or trains intuition, or points to patterns that might hold approximately in nature. Only rarely does biological theory provide the essentially exact results that physicists are accustomed to. (And in biology approximate results, or even rules of thumb, are often more useful than exact results.) This kind of broad-stroke theory doesn&rsquot provide much help with a problem as specific as the coding question.

A rough analogy captures these kinds of concerns. Mathematical theory might tell you something interesting and general about combination locks: for example, that they should require a sequence of three or more numbers to prevent a would-be thief from opening them in a few random tries. But place a particular combination lock before a theorist and he&rsquos probably no better than the rest of us at opening it.

Finally, and perhaps most important, Life&rsquos Greatest Secret highlights the power of the beautiful experiment in science. Though Cobb pays less attention to this subject than he might have, the period of scientific history that he surveys was the golden age of the beautiful experiment in biology. Biologists of the time&mdashincluding Nirenberg with his UUU, Crick and Brenner with their triplet code work, and others including Matthew Meselson, Franklin Stahl, and Joshua Lederberg&mdashwere masters of the sort of experiment that, through some breathtakingly simple manipulation, allowed a decisive or nearly decisive solution to what previously seemed a hopelessly complex problem. Such experiments represent a species of intellectual art that is little appreciated outside a narrow circle of scientists.


Kyk die video: Decode from DNA to mRNA to tRNA to amino acids (Oktober 2022).