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Next to the directed evolution of existing ribozymes <i>in vitro</i> selection methods<x-ref>bartel1993</x-ref> were developed to select nucleic acids with new catalytic activities from a random pool. Catalytic RNA of versatile functions arose from these methods. For instance Seelig and Jäschke selected a diels alderase ribozyme catalyzing the Diels-Alder reaction leading to the formation of a carbon-carbon bond.<x-ref>seelig1999</x-ref> Not only can <i>in vitro</i> selection methods be applied to reveal new catalytic RNA but also to select aptamers.
 
Next to the directed evolution of existing ribozymes <i>in vitro</i> selection methods<x-ref>bartel1993</x-ref> were developed to select nucleic acids with new catalytic activities from a random pool. Catalytic RNA of versatile functions arose from these methods. For instance Seelig and Jäschke selected a diels alderase ribozyme catalyzing the Diels-Alder reaction leading to the formation of a carbon-carbon bond.<x-ref>seelig1999</x-ref> Not only can <i>in vitro</i> selection methods be applied to reveal new catalytic RNA but also to select aptamers.
 
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Revision as of 17:24, 18 September 2015

Catalytic RNA – Ribozymes

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In 1982 the first catalytic RNA (Ribozyme): a self-spicing intron from Tetrahymena pre-rRNA was described.cech1981Kruger1982 Shortly after the discovery of these self-spicing introns further ribozymes were identified in different organisms. Amongst which the hammerhead ribozyme (HHR) is a very prominent example (Fig.1) that has been employed by many laboratories since its discovery in 1986.Prody1986Forster1987 This ribozyme has the capability of self-cleavage. A HHR consists of three stems of which one is open. The complementary region of this open stem can be designed so that the HHR cleaves off a customized sequence without leaving a scarMeyer2014. (Click for design guide) This makes it a valuable tool in the work with functional RNA as it leaves defined 5’- or 3’-ends after cleavage that are essential for some applications.balke2014

Another ribozyme that is related to the HHR is the hepatitis δ virus ribozyme (HDV).Sharmeen1988 In contrast to the HHR it cleaves itself off a RNA at a specific position without a recognition site and without leaving a scar. This advantage of being independent from an upstream sequence makes it widely applicable and thus is used in our new BBF RFC 110 that aims to simplify and standardize the use of functional RNA.

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Both of the mentioned types of ribozymes are found in satellite RNA of plant originSerganov2007 as does the so called hairpin ribozyme (Fig. 2). It is a ribozyme capable of cleaving and ligating a RNA complementary to itself.Buzayan1986 While researchers were looking for naturally occurring ribozymes, they developed methods to in vitro evolve the function of existing ribozymes and to discover new ones from random pools.beaudry1992 This way scientists were able to establish catalytic systems with new function. For example Joyce developed a self-replicating system consisting of two hairpin-ribozyme-derived parts that replicate each other.lam2009lincoln2009 Our idea of working with functional nucleic acids originated from this system. We were fascinated by the vast variety of processes that they can perform and started digging deeper into the potential of nucleic acids as tools. During this process we came across interesting systems amongst which we found the twin ribozyme (Fig. 3). Another famous hairpin-ribozyme-derived functional nucleic acid developed by Müller.schmidt2000 It is able to specifically cleave a RNA at two designed specific positions and afterwards re-ligate it. Balke Balke2014 applied the twin ribozyme as tool to edit mRNA in vitro.balke2014 In our project we want to use this system to specifically repair the mutation in a mRNA leading to the malfunction of a protein. The function of the protein is restored by inserting the missing bases and thus the patients symptoms disappear.

Next to the directed evolution of existing ribozymes in vitro selection methodsbartel1993 were developed to select nucleic acids with new catalytic activities from a random pool. Catalytic RNA of versatile functions arose from these methods. For instance Seelig and Jäschke selected a diels alderase ribozyme catalyzing the Diels-Alder reaction leading to the formation of a carbon-carbon bond.seelig1999 Not only can in vitro selection methods be applied to reveal new catalytic RNA but also to select aptamers.

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