A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

A Processive Protein Chimera Introduces Mutations across Defined DNA Regions <i>In Vivo</i>

Laboratory time scale evolution <i>in vivo</i> relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted <i>in vivo</i> mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for <i>in vivo</i> targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of <i>in vivo</i> evolution experiments

Ribosomal Synthesis of Macrocyclic Peptides <i>in Vitro</i> and <i>in Vivo</i> Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner <i>in vitro</i> or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low-micromolar streptavidin binder (<i>K</i>_D = 1.1 μM) from a library of cyclic peptides produced in Escherichia coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles

Comprehensive synthesis of various functionalized graphene nanoribbons

The contemporary semiconductor industry is experiencing problems with the miniaturization and efficiency of silicon-based components. After its first successful isolation in 2004, graphene has been widely researched due to the potential of surpassing silicon as the ubiquitous semiconductor material. The unprecedented electric conduction properties combined with possibilities of controlling the graphene fragment edges with atomic precision make it a true nano-scale building material that might revolutionize the whole logic circuit and semiconductor industries. Graphene is the carbon allotrope of single atomic layer of carbon atoms arranged into a hexagonally symmetrical honeycomb structure. Due to the confinement into a singular layer and the ultimate symmetry of the lattice, graphene exhibits highly unusually hybridized energy bands, which are the source of its coveted electromagnetic and mechanical properties. These properties can be tuned in a versatile manner by patterning graphene in nanometer scale. Especially the symmetric and well-defined graphene nanoribbons (GNRs) are of high interest, and their production has been under increasing research. The aim of this thesis was to assess various methods capable of producing nano-scale functionalized graphene structures. The most advanced contemporary method for this is the surfaceassisted self-assembly (SASA) of intelligently designed precursor molecules on transition metal surfaces. Five batches of different target molecules were synthesized in order to be used as precursor materials for GNR formation through the SASA approach. The synthesis details of these molecules have been presented in the experimental section of this work. The SASA approach is currently the only synthesis method capable of providing atomic scale control over the graphene domain edges. Other graphene synthesis methods include exfoliation from graphite, unzipping carbon nanotubes and epitaxial film synthesis from simple hydrocarbon gasses. The quality of graphene is highly affected by the synthesis transition metal catalyst, and the properties exhibited by graphene domains are directly influenced by the substrate. It is therefore of high importance to be aware of the properties and interactions of various substrate materials. Decoupling the graphene fragments from unfit surfaces and applying them on optimal substrates is a necessary step of the synthesis. Hexagonal boron nitride (hBN) is currently the best known substrate for graphene-based devices. While the exceptional nature of graphene has already been demonstrated by producing highly effective experimental devices, the challenges associated with scale-up and mass-production of well-defined graphene structures remain currently unsolved.Nykyaikaisessa puolijohdeteollisuudessa pii-peräisten komponenttien tehostukseen ja miniatyrisointiin liittyviä ongelmia on yritetty ratkaista etsimällä vaihtoehtoisia komponenttimateriaaleja. Grafeenia on tutkittu runsaasti vuoden 2004 jälkeen, jolloin ensimmäinen onnistunut yksikerroksisen grafeenin eristystapa esitettiin. Grafeenin erityislaatuiset sähkönjohtokykyyn liittyvät ominaisuudet yhdistettynä grafeenisaarekkeiden mahdolliseen atomitason reunakontrolliin tekevät siitä potentiaalisesti mullistavan nano-mittakaavan logiikkapiiri- ja puolijohdemateriaalin. Grafeeni on hiilen allotrooppi, jossa hiiliatomit järjestäytyvät yksikerroksiseksi, säännölliseksi, kuusikulmaiseksi hilaksi. Korkeasta symmetriasta ja yhden atomin paksuudesta johtuen grafeenin energiatasot ovat hybridisoituneet hyvin epätavallisen muotoisesti, mistä johtuen mm. sen sähkönjohtokyky on ilmiömäinen. Näiden energiatasojen (ja siten sähkömagneettisien ominaisuuksien) järjestäytymiseen voidaan vaikuttaa monipuolisesti leikkaamalla grafeenia nanometrien mittakaavassa. Erityisesti symmetristen ja atomitasolla kontrolloitujen grafeeninauhojen (GNR) onnistunut ja laajamittainen synteesi on suuresti kiinnostava ja tutkittu aihe. Tämän työn tarkoituksena oli perehtyä mahdollisiin keinoihin valmistaa nanometrien mittaisia, funktionalisoituja grafeenirakenteita. Edistynein nykyaikainen menetelmä tähän tarkoitukseen on ennalta suunniteltujen lähtöaineiden hallittu ja katalysoitu terminen hajottaminen ja yhdistyminen laadukkailla siirtymämetallipinnoilla (SASA). Tähän tarkoitukseen valmistettiin viisi eri tuotemolekyylierää, joiden synteesireitit on esitelty työn tutkimusosassa. Grafeenirakenteiden reunojen atomitason kontrolli ei ole toistaiseksi mahdollista muilla menetelmillä, joilla on puolestaan muita etuja käytännön grafeenikomponenttien valmistusta ajatellen. Näitä menetelmiä ovat mm. grafeenin mekaaninen kuoriminen grafiitista, nanoputkien kemiallinen aukaiseminen, sekä epitaksiaalisten grafeenifilmien valmistus yksinkertaisista hiilivedyistä. Koska grafeenin laatu ja havaittavat ominaisuudet riippuvat suuresti synteesissä käytetystä siirtymämetallista sekä pinnasta, jonka päälle se on sittemmin asetettu, on pintamateriaalien aiheuttamien interaktioiden tunteminen tärkeää. Synteesivaiheessa katalyyttisten ominaisuuksiensa takia käytetyt siirtymämetallit eivät sovellu alustoiksi mahdollisille grafeenikomponenteille. Tästä syystä grafeenin siirtomahdollisuudet tulee ottaa synteesissä huomioon. Heksagonaalinen boorinitridi (hBN) on tähän mennessä paras tunnettu pintamateriaali grafeenille. Tähän mennessä on jo näytetty toteen grafeenin erinomaisuus kokeellisten komponenttien muodossa, mutta massatuotantoon ja funktionalisoitujen grafeenirakenteiden ongelmattomaan synteesiin liittyvät haasteet ovat vielä toistaiseksi ratkaisematta