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Red light-sensing phytochrome-DNA conjugates for nanotechnology applications

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Authors
Kettunen, Iida
Date
2020
Discipline
Solu- ja molekyylibiologiaCell and molecular biology
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DNA-nanoteknologiassa on kehitetty paljon erilaisia DNA-origamiteknologiaa hyödyntäviä nanokoneita viime vuosikymmenien aikana. Kuitenkaan punavalo-ohjattavia DNA-koneita, joita voitaisiin soveltaa biologisiin käyttökohteisiin, ei ole valmistettu. Tätä varten DNA-koneet pitäisi funktionalisoida punavaloa aistivilla molekyyleillä. Fytokromit ovat punaista ja pitkäaaltoista punaista valoa aistivia valoreseptoreita, joita tavataan luonnossa kasveilla, bakteereilla, sienillä ja levillä. Valoaistimuksen seurauksena ne muuttavat konformaatiotaan kahden eri tilan välillä. Jotkin fytokromit, kuten Agrobacterium fytokromi 1 (Agp1), muuttavat myös kvaternäärirakennettaan valon seurauksena. Tämän työn tavoite oli valmistaa Agp1-DNA-konjugaatteja ja karakterisoida niiden toiminnallisuutta, jotta niitä voitaisiin hyödyntää punavalokontrolloitavien DNA-koneiden komponentteina. Agp1:stä suunniteltiin ja tuotettiin kaksi pintakysteiineillä varusteltua mutanttia (E379C ja D367C), jotka konjugoitiin DNA-oligonukleotidien kanssa käyttäen dibentso-oktyyli-maleimidia verkkosidoksena. Konjugaattien valokonversiota karakterisoitiin ultravioletti- ja näkyvän valon spektroskopialla ja kvaternäärirakennetta kokoerottelukromatografialla. DNA:n kiinnittäminen ei häirinnyt fytokromien valokonversiota ja Agp1 osoitti merkkejä punavaloriippuvaisesta dimerisaatiosta. E379C-mutantti muodosti huomattavasti enemmän konjugaatteja ja oli siten parempi vaihtoehto DNA-nanokoneille. Tämä työ osoitti, kuinka fytokromeja voidaan muokata, jotta niitä voitaisiin käyttää dynaamisina kytkiminä DNA-nanoteknologian sovelluksissa mahdollistaen uudenlaisten, reversiibelien ja punavalo-ohjattavien DNA-koneiden kehittämisen. ...
 
DNA nanotechnology has boosted during the past few decades developing a broad range of nanodevices that exploit DNA origami. However, red light-controllable DNA devices that could allow controllability in biological environments have not yet been demonstrated. For this, DNA nanostructures should be functionalized with red light-responding molecules. Phytochromes are red and far-red light-sensing photoreceptors that are naturally encountered with plants, bacteria, fungi and algae. They convert between two conformational states in response to red light illumination. Some phytochromes, such as Agrobacterium phytochrome 1 (Agp1), additionally change their quaternary structure in red light-dependent manner. The aim of this thesis was to conjugate Agp1 with DNA oligonucleotides, and characterize their functionality for their use in red light-controllable DNA nanodevices. Two Agp1 mutants with surface cysteines (E379C and D367C) were produced and conjugated with DNA oligonucleotides using dibenzocyclooctyl-maleimide as a crosslinker. The photoconversion of the conjugates was characterized with ultraviolet-visible light spectroscopy, and the red light-induced dimerization was studied with size exclusion chromatography. These experiments revealed that the photoconversion was unaffected by the DNA attachment, and suggested Agp1 dimerization in consequence to red light stimuli. The E379C mutant was a better candidate for DNA nanodevices, as it had a significantly higher conjugation yield. This study demonstrated how phytochromes can be modified in order to use them as dynamic switches for DNA nanodevices, and thus enabling the preparation of novel, reversible and red light-controllable DNA machines. ...
 
Keywords
Agp1 protein-DNA conjugation quaternary structure size exclusion chromatography kromatografia nanotekniikka chromatography nanotechnology
URI

http://urn.fi/URN:NBN:fi:jyu-202007285404

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