The science of biotechnology
Ronald P. Evens in Biotechnology, 2020
Ribozymes are RNA molecules comprising sequences of nucleic acids that possess enzymatic catalytic properties and bind to specific sites in DNA or RNA and cleave the chain. A ribozyme will have subunits responsible for the binding function and subunits responsible for enzyme function. They generally have the following desirable traits: specificity in targeting, cleavage of target RNA, small size amenable to formulation and dosing, and multiple turnover (one molecule binds and acts and then moves on to next molecule and repeats its function). However, challenges are manifold; for example, the need for cell insertion (transfection), nuclease protection in the blood, and chaperone proteins for movement in cell cytoplasm. No products have yet been approved. Aptamers are small oligonucleotide molecules that bind to proteins to disrupt disease pathogenesis. Their benefits are small molecular size, specificity toward target protein, and low immunogenicity; however, limitations are nuclease susceptibility, short systemic half-lives, and possibly limited affinity to targets. Proof of principle has been achieved as one aptamer has been approved for use, a pegylated conjugate of an oligonucleotide for wet acute macular degeneration, pegaptanib (Macugen®). The pegylation protects the molecule from lysis from nucleases, and offers a longer serum half-life and a measure of immunity.
Beyond Enzyme Kinetics
Clive R. Bagshaw in Biomolecular Kinetics, 2017
In addition to the well-known helical duplex structures, nucleic acids and RNA, in particular, can form higher-order (tertiary) structures. The kinetics of formation of such states is analogous to protein folding (Section 5.1), with kinetics that may appear relatively simple, but encompass a large number of microstates [218,219]. Some RNA molecules, known as riboswitches, change conformation on binding small metabolites and constitute important cellular control mechanisms [205,220–222]. In addition, some RNA molecules show catalytic activity, a finding that overturned the idea that all enzymes were proteins [223]. However, such ribozymes catalyze a limited repertoire of reactions (typically cleavage of the ribophosphate backbone of a nucleic acid). Often they only undergo a single turnover and hence are not catalysts in the conventional sense [224]. Furthermore, reaction times are on the order of minutes rather than milliseconds typical of proteinaceous enzymes [225]. This characteristic allows transient kinetic reactions to be followed by manual quenching and analysis by gel electrophoresis [224].
Evolution
Paul Pumpens in Single-Stranded RNA Phages, 2020
The history and progress of the scientific and practical applications of the molecular colonies was analyzed in detail by Chetverin and Chetverina (2007). In particular, they noticed that Mitra and Church (1999) published a PCR version of the molecular colony technique under the name of polony (polymerase colony) technology. Next, they noticed that Szostak (1999) was the first to indicate that molecular colonies, analogous to those formed when RNA was amplified in agarose with Qβ replicase, could have represented a precellular variant of compartmentalization in the RNA World. In this case, the compartmentalization was provided not by an envelope but by a relatively low diffusion rate of macromolecules in a porous matrix as compared with the diffusion rate of low molecular mass substances. According to Szostak, the RNA colonies could be formed in moist clay and other porous minerals (cited by Chetverin and Chetverina 2007). This idea was further developed by Alexander Spirin (2002, 2005a,b), who postulated that the mixed colonies comprising the following three species of macromolecules could be the first individuals in the RNA World: (i) ligand-binding RNA molecules for selective adsorption and accumulation of necessary substances from the environment; (ii) a set of ribozymes catalyzing the metabolic reactions of nucleotide synthesis; and (iii) a ribozyme catalyzing complementary replication of all RNA molecules of the colony.
The discovery and development of RNA-based therapies for treatment of HIV-1 infection
Published in Expert Opinion on Drug Discovery, 2023
Michelle J Chen, Anne Gatignol, Robert J. Scarborough
Ribozymes are RNAs that catalyze biochemical reactions. The first ribozyme was identified in self-splicing introns, where the RNA catalyzes both cleavage and ligation reactions that result in the excision of the intron from the transcript [38]. Subsequently, it was shown that RNA is the catalytic moiety in RNase P complexes that cleave pre-transfer (t)RNAs [39] and in ribosomes, where ribosomal RNA is responsible for catalyzing the linkage of amino acids to form proteins [40]. The most diverse group of ribozymes are the small, naturally occurring, self-cleaving ribozymes from which most ribozyme therapies have been derived [29,41]. Although these ribozymes catalyze self-cleavage reactions, they can be easily modified to cleave in trans and designed to target an RNA through complementary base pairing. An advantage of small self-cleaving ribozyme motifs is that they do not require cellular proteins to catalyze target cleavage, limiting their ability to disturb cellular physiology. Examples of trans-cleaving ribozymes based on these motifs are shown in Figure 1.
Challenges with the discovery of RNA-based therapeutics for flaviviruses
Published in Expert Opinion on Drug Discovery, 2023
Mei-Yue Wang, Rong Zhao, Yu-Lan Wang, De-Ping Wang, Ji-Min Cao
In addition, there are some ribozyme-based strategies against flaviviruses. Ribozymes are small RNA molecules with catalytic activity and can target the particular site of RNA to cleave it. For instance, Nawtaisong et al. [36] constructed 14 hammerhead ribozymes and tested the effectiveness of these ribozymes in the suppression of DENV-2 replication in lentivirus-transduced mosquito cells. The ribozymes were expressed using pan retroviral vectors and controlled by Aedes aegypti tRNAval promoter. In lentivirus-transduced C6/36 mosquito cells, 100-fold suppression of virus replication could be observed. Another study designed the group I trans-splicing introns (a type of ribozyme) to target the conserved 5’-3’ cyclization sequence (CS) region which is common to all four DENV serotypes (DENV-1–4) [37]. The examples are presented in Table 1.
Nucleic acid therapeutics: a focus on the development of aptamers
Published in Expert Opinion on Drug Discovery, 2021
Swati Jain, Jaskirat Kaur, Shivcharan Prasad, Ipsita Roy
Ribozymes are small RNA molecules of about 50–150 nucleotides that perform site-specific self-cleavage on RNA [45,46]. An example of glmS ribozyme has been described above [43]. The catalytic function of RNA was first demonstrated in the laboratory of Tom Cech, where ribosomes were shown to function as ribozymes [47], and by Sidney Altman, whose group showed that the catalytic activity of bacterial RNase P resided in a ribonucleotide [48]. Like riboswitches, ribozymes can be of natural or synthetic origin. In the latter case, their properties can be tailored to requirement [49]. Ribozymes have been used to validate disease-related genes as potential targets for new therapeutic interventions [50]. Because of their high specificity at the pre-translation stage, ribozymes can play important roles in therapeutics and diagnostics [51]. Another molecule similar to ribozyme is DNAzyme. DNAzymes or deoxyribozymes are single-stranded DNA molecules of synthetic origin which possess catalytic activity. Since the first report of the selection of a DNAzyme from a randomized library which could carry out Pb2+-dependent hydrolysis of a specific ribonucleotide phosphate bond [52], the field has expanded to include applications in diverse areas where the more fragile enzymes or ribozymes are not suitable [53,54,55]. More importantly, DNAzymes have been shown to be important in designing therapeutic strategies [5]. Their application in oncology was demonstrated by designing a DNAzyme targeting vascular endothelial growth factor receptor-1 (VEGFR-1) in a rabbit liver cancer model [56]. Dynamic contrast enhanced magnetic resonance imaging showed positive effect on tumor vasculature via downregulation of VEGFR-1 expression, with concomitant improvement in microvascular permeability parameters.
Related Knowledge Centers
- Catalysis
- DNA
- Enzyme
- Gene Expression
- Ribosome
- Rna
- Rna Splicing
- Rna World
- Directed Evolution
- Translation