Ribozymes novel

Since the discovery that RNA molecules can possess catalytic activities, ribozymes have become a fascinating field both for academic researchers and the pharmaceutical industry. In this review, we emphasize the latest progress made in structure determination of ribozymes as well as the generation of DNA and RNA enzymes with novel catalytic properties by combinatorial approaches. 

One of the most interesting ribozymes isolated by in vitro selection techniques is a novel catalytic RNA that promoted the formation of a glycosidic bond... 

A novel finding related to the mechanism of catalysis by the genomic HDV ribozyme is that the pKa of C75 is perturbed to neutrality in the ri-bozyme-substrate complex and, more importantly, that C75 acts as a general acid catalyst in combination with a metal hydroxide which acts as a general base catalyst (Fig. 9A) [105]. The discovery of this phenomenon provided the first direct proof that a nucleobase can act as an acid/base catalyst in RNA. As a result, as shown by the solid curve in Fig. 9B, the curve that represents the dependence on the pH of the self-cleavage of the precursor genomic HDV ribozyme has a slope of unity at pH values that are below 7 (the activity increases linearly as the pH increases, with a slope of +1). Then, at higher pH values, the observed rate constant is not affected by the pH. 

Figure 11.4 The hypothetical pathway for the transformation of a simple RNA cell into a minimal DNA/protein cell. At the first step, the cell contains two ribozymes, Rib-1 and Rib-2 Rib-1 is a RNA replicase capable of reproducing itself and making copies of Rib-2, a ribozyme capable of synthesizing the cell membrane by converting precursor A to surfactant S. During replication, Rib-1 is capable of evolving into novel ribozymes that make the peptide bond (Rib-3) or DNA (Rib-4). In this illustration, these two mutations are assumed to take place in different compartments, which then fuse with each other to yield a protein/DNA minimal cell. Of course, a scheme can be proposed in which both Rib-3 and Rib-4 are generated in the same compartment. (Modified fromLuisi et al., 2002.)...

Novel biocatalysts, such as catalytic antibodies or ribozymes, that have recently been... 

Chowrira, B. M., A. Berzal-Herranz, and J. M. Burke, Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature 354 320-323, 1991. In some cases the guanine amino group serves a catalytic role in selfsplicing. 

Kuwabara, T., Warashina, M., Tanabe, T., Tani, K., Asano, S. and Taira, K. (1998) A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo. Mol. Cell, 2, 617-627. 

Finally, novel nucleic acid catalysts have also been selected from random sequence pools (reviewed in Ref. 19). Joyce and co-workers have manipulated the function of the Group I self-splicing ribozyme, selecting variants that can utilize calcium or cleave DNA from partially randomized pools [20,21], Lorsch and Szostak [22] selected a polynucleotide kinase ribozyme from a completely random sequence pool that flanked a previously selected ATP binding site. Many of the novel ribozymes can catalyze reactions that are relevant to protein biosynthesis, bolstering arguments that translation may have arisen in a putative RNA world. For example, Lohse and Szostak [23] have selected ribozymes that can carry out an acyl transfer reaction, while Illangasekare et al. [24] have isolated a... 

In this section is an overview of some historical aspects of enzyme studies with special emphasis on new methods of purification, structure determination, and research on the reaction mechanism of an enzyme. Enzyme application for medical and industrial use and development of novel enzymes such as catalytic RNA (ribozyme) and catalytic antibody (abzymes) are also briefly described. 

Design of RNA molecules with novel catalytic functions called ribozymes (ribonucleotide enzymes) started out from the reprogramming of naturally occurring molecules to accept unnatural substrates [32, 33] A specific RNA cleaving ribozyme, a class I (self-splicing) intron, was modified through variation and selection until it operated efficiently on DNA. The evolutionary path of such a transformation of catalytic activity has been recorded in molecular detail [34]. The basic problem in the evolutionary design of new catalysts is the availability of appropriate analytical tools for the detec-... 

Oligonucleotide libraries are commonly used either to encode biosynthetic display peptide libraries (Section 11.1) or to screen for novel nucleotide ligands (aptamer) or catalytic activities (ribozymes). They are produced using high-quality automated ON SP protocols seen in Section 2.2, but their amplification and selection are performed via biological protocols (see Sections 4.2.4 and 11.2). 

The results from this and another similar study (235) could be the precursors to the identification of novel, potent ribozymes able to catalyze organic reactions and to... 

B.M. Chowrira, A. Berzalherranz, J.M. Burke, Novel Guanosine Requirement for Catalysis by the Hairpin Ribozyme , Nature, 354, 320 (1991)... 

Nucleic acid selection methods have also been exploited for the development of novel RNA enzymes or ribozymes (58). An m-vitro-selected RNA that contains the modified nucleotide 5-(4-pyridylmethyl)-uridine (Table 1) can catalyze carbon-carbon bond formation in a Diels-Alder cycloaddition, with an 800-fold rate acceleration compared with a random RNA (49). Modified RNAs that contain the same uridine modification have also been selected to mediate metal-metal bond formation in the synthesis of palladium nanoparticles (59). Modified RNAs are likely to have many other applications as novel ribozymes that catalyze important biological reactions or can be used to create novel materials. 

RNA is chemically very similar to DNA but differs in important ways. The sugar miit is ribose with an added hydroxyl group at the 2 position, and the methylated pyrim-idine uracil (U) replaces thymine. RNA exists in various functional forms but typically as a single-stranded polymer that is much shorter than DNA and that has an irregular three-dimensional structure. Research from recent years has revealed that RNA conformations are not random structures and the folding mechanism of RNA molecules is complex. The secondary structure adopted by an RNA molecule is to a large extent related to its nucleotide sequence. The secondary structure for particular RNA sequences can be as regular as the secondary structure of a protein. It is now known that RNA molecules can further interact to form complex tertiary structures, which are intimately related to novel functions of RNA, such as the catalytic activity of ribozymes, ... 

After ribozyme (100-mer) catalyzing Diels-Alder reaction was discovered in 1997 using the unique selection system, small Diels-Alder ribozyme (38-mer) with remarkable catalytic efficiency (20 000 relative to the uncatalyzed reaction) was developed. Recently, the novel ribozyme that catalyzed [4 - - 2] cycloaddition between tethered diene and biotinylated maleimide (Scheme 20) and the crystal structure of Diels-Alder ribozyme in the unbound form and in complex with a reaction product has been solved. ... 

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