Gerhard Steger - DNAzymes : Methods and Protocols

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Ribozymesenzymes consisting of only RNAwere well established in 1990: Thomas Czech and coworkers had established the self-splicing of Tetrahymena group I intron [1, 2], Sidney Altman and coworkers had described the ribozyme nature of RNAse P cleaving off a precursor of tRNA [3, 4], George Bruening and coworkers had detected the self-cleavage activity of satellite RNAs [5], and Robert Symons and coworkers had described the self-cleavage activity of viroids from family Avsunviroidae [6]. At this time, most scientists expected that such ribozyme activity would be restricted to RNA because DNA misses several features that were thought to be essential for catalytic activity: DNA occurs mostly not single-stranded and thus rarely forms complex tertiary structures, and DNA lacks the 2-OH group of RNA. Thus, the first publication on a DNAzymeDNA with enzymatic activityby Ronald Breaker and Gerald Joyce [7] in 1994 came as a big surprise. Today, a broad variety of DNAzymes with different catalytic functions are known [8], including RNA cleavage [7], peptide modification [9, 10], phosphorylation [11], thymine dimer photoreversion [12], peroxidation [13], and DNA ligation [14]. Schematic representations of four selected DNAzymes are shown in Fig..

The DNAzyme described by Breaker and Joyce in 1994 and most of the many DNAzymes described afterwards were found by in vitro selection starting from a random pool of DNA molecules, called systematic evolution of ligands by exponential enrichment (SELEX) [16]. The principle of SELEX to obtain molecule(s) with a desired function is based on the assumption that a three-dimensional structure, which is required for function, can be formed by many different single-stranded sequences. In theory, a random pool of sequences with 15 up to 50 nucleotides in length and the four different nucleotides comprises 415 1.2 1018 up to 450 1.3 1030 different sequences, covering all possible structures within the limits of the sequence length; in practice this number of random sequences is limited to about 1014 to 1015 sequences due to additionally required constant sequence parts, for example for amplification, hybridization, and fixation. One key step in the selection process is the separation of nucleic acids with the desired functionality from those which lack this property and therefore should be excluded from the pool in the next selection round. The other key step is amplification of the relatively low amount of recovered nucleic acids with the desired functionality after each selection round by polymerase chain reaction (PCR) [17, 18], which requires fixed primer binding sites at the ends of the random sequence part.