Cleaving RNA

Although, as stated above, we wiU mostly focus on hydrolytic systems it is worth discussing oxidation catalysts briefly [8]. Probably the best known of these systems is exemphfied by the antitumor antibiotics belonging to the family of bleomycins (Fig. 6.1) [9]. These molecules may be included in the hst of peptide-based catalysts because of the presence of a small peptide which is involved both in the coordination to the metal ion (essential co-factor for the catalyst) and as a tether for a bisthiazole moiety that ensures interaction with DNA. It has recently been reported that bleomycins will also cleave RNA [10]. With these antibiotics DNA cleavage is known to be selective, preferentially occurring at 5 -GpC-3 and 5 -GpT-3 sequences, and results from metal-dependent oxidation [11]. Thus it is not a cleavage that occurs at the level of a P-O bond as expected for a non-hydrolytic mechanism. 

Smaller self-cleaving RNAs have been found among plant viruses and viroids. Many of them have a common catalytic core which can be converted into 30- to 40-nucleotide ribozymes. Only 17 nucleotides and three hydrogen-bonded helical stems are required to form the self-cleaving "hammerhead" domain, which has a structural similarity to the catalytic core of the Tetrahymena ribozyme. The hammerhead ribozymes (Fig. 12-27) represent one form of small ribozyme.793-797 Another is the hairpin ribozyme shown in Fig. 

In an intact viral self-cleaving RNA the entire catalytic center is formed from a single strand. Stems... 

Some newly synthesized RNA transcripts are the functional species, whereas others must be modified or processed into the mature functional species. Modifying enzymes add nucleotides to the 5 or 3 ends or alter bases within the RNA, such as by methylation of specific residues. Specific processing enzymes cleave RNA internally, splice together noncontiguous regions of a transcript, or remove nucleotides from the 5 or 3 ends. 

The most common method employs the substitution of all pyrimidine nucleotides by their 2, -NH2- or 2 -F-analogs [31]. These confer increased stability to the RNA, because most ribonucleases need the 2 -OH for cleaving RNA. Substitution of only the pyrimidine nucleotides increases the half-life of RNA from seconds to days [28]. Such -modified RNAs are suitable for the detection of analytes in biological samples like blood, serum, or urine. 

FEN-1 is a 43 kDa metallonuclease, which cleaves 5 single-strand DNA flap structures and possesses the capacity to digest DNA ends in the 5 to 3 direction (Harrington and Lieber, 1994). FEN-1 also cleaves RNA flaps and has a 5 to 3 exonuclease activity of double-stranded DNA or RNA annealed with DNA (Huang et al., 1996). 

Silverman, S. K. (2005). In vitro selection, characterization, and application of deoxyribozymes that cleave RNA. Nucleic Acids Res. 33, 6151—6163. 

Ribonuclease A is a member of a group of enzymes that cleave RNA using general acid-base catalysis without a metal ion in the enzyme. In ribonuclease A, such catalysis is performed by two imidazoles of histidine units, one as the free base (Im) and the other, protonated, as the acid (ImH+). To mimic this in an artificial enzyme, we prepared (3-cyclodextrin bis-imidazoles 41 [124]. The first one was a mixture of the... 

Figure 4 The enzyme ribonuclease A cleaves RNA using the imidazole group of histidine 12 as a base and the imidazolium group of histidine 119 as an acid in a simultaneous two-proton transfer process. Studies discussed in this article indicate that the process is not as simple as shown.

The hepatitis delta virus (HDV) ribozyme is a member of the class of small ribozymes and functions as a self-cleaving RNA sequence critical to the replication of the virus RNA genome (1, 8, 40). HDV ribozymes are proposed to employ several catalytic strategies that include an important example of general acid/base catalysis that involves a specific cytosine residue in the active site. Indeed, a milestone in our understanding of RNA catalysis was the observation that HDV and other small ribozymes could function in the absence of divalent metal ion cofactors, provided that high (molar) concentrations of monovalent ions are present (41, 42). These high monovalent ion concentrations are believed to stabilize the active RNA conformation, which implies that the primary role of divalent metal ions is in structural stabilization (42). 

Phosphorothioate-modified DNA aptamers have been selected for binding to NF-kB, ° and phosphoramidate ODNs for binding to TAR to inhibit Tat-mediated transcription. Deoxyribozymes that cleave RNA have been modified with 3 -3 inverted linkages, phosphorothioate linkages, 2 0-methyl sugars and LNA in an attempt to enhance stability and cleavage activity. " ... 

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