Overview of Ribozymes

Ribozymes are RNA molecules that can catalyze specific biochemical reactions, such as RNA splicing in gene expression. They are also known as ribonucleic acid enzymes or catalytic RNAs. Their function is analogous to that of protein enzymes, which are the most common biological catalysts. Ribozymes were discovered in Tom Cech's laboratory at the University of Colorado in 1982. This discovery revealed that RNA can act as both genetic material (like DNA) and a biological catalyst (like protein enzymes), and supported the RNA world hypothesis, which proposes that RNA may have played a key role in the evolution of prebiotic self-replicating systems.

Introduction of Ribozymes

Before ribozymes were discovered, it was believed that only enzymes, which are proteins that catalyze biochemical reactions, could act as biological catalysts. The idea that RNA could act as a catalyst was proposed based on the discovery that RNA can form complex secondary structures. The first ribozyme to be discovered was spliceosomes, or self-splicing introns, in the ribosomal rRNA genes of the ciliated protozoan Tetrahymena thermophila. Later, ribozymes in the bacteria were discovered. Ribonuclease P, a bacterial enzyme, comprises RNA and protein, where RNA exhibits catalytic activity. Ribonuclease P is involved in the sizing of large RNA precursors into smaller RNAs (RNA processing). In addition, several other types of self-cleaving or catalytic RNA molecules have been identified by different researchers. Many ribozymes have a hairpin- or hammerhead-shaped active site and a distinctive secondary structure that enables them to cleave other RNA molecules at specific sequences. It is feasible to design ribozymes that will selectively cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. As an example, a ribozyme that can cleave the RNA of human immunodeficiency viruses (HIV) has been engineered. Such a ribozyme could confer resistance to HIV infection by destroying the RNA genome of the incoming virus particles in the cell.

Classification of Ribozymes

Ribozymes are classified into two classes based on their size and reaction mechanisms. The large ribozymes include RNase P and the group I and group II introns. These molecules vary in size from a few hundred to about 3,000 nucleotides. They catalyze reactions that generate reaction intermediates and products with 3' hydroxyls and 5' phosphates. The small catalytic RNAs range in size from ~35 to ~155 nucleotides and include the hammerhead, the hairpin (or paperclip), hepatitis delta, and VS RNA. They use the 2' hydroxyl of the ribose sugar as a nucleophile, and they generate products with a 2',3'-cyclic phosphate and a 5' hydroxyl. The relationship between the size and the reaction mechanism of these molecules has raised intriguing questions about their origins and evolution. The large size and reaction mechanism of the large ribozymes may enable them to align distant elements of the substrate for catalysis.

Structures and Mechanisms of Ribozymes

Despite having only four options for each monomer unit (nucleotides), unlike 20 amino acid side chains in proteins, ribozymes exhibit diverse structures and mechanisms.

Figure 1. General Mechanism of Ribozyme.

In many cases, they can emulate the mechanism used by their protein counterparts. For instance, in self-cleaving ribozyme RNAs, an in-line SN2 reaction is performed using the 2' hydroxyl group as a nucleophile that attacks the bridging phosphate and causes the 5' oxygen of the N+1 base to act as a leaving group. Likewise, RNase A, a protein enzyme that catalyzes the same reaction, employs a histidine and a lysine residue to coordinate and act as a base that attacks the phosphate backbone. Like many protein enzymes, metal binding is also essential for the function of many ribozymes. Often these interactions involve both the phosphate backbone and the base of the nucleotide, resulting in drastic conformational changes.

Figure 2. Small self-cleaving ribozymes. (Ferré-D'Amaré AR. 2010)

There are two classes of mechanisms for the cleavage of a phosphodiester bond in the presence of metal ions. In the first class, the internal 2'-OH group of the RNA acts as a nucleophile and displaces the phosphate oxygen in an SN2 reaction. The metal ions facilitate this reaction by coordinating the phosphate oxygen before the cleavage and stabilizing the oxyanion after the cleavage. In the second class, the nucleophile is a water molecule or an exogenous hydroxyl group, rather than the RNA itself, and it also undergoes an SN2 reaction with the phosphate center. The smallest ribozyme is UUU, which can promote the cleavage between G and A of the GAAA tetranucleotide via the first mechanism in the presence of Mn²⁺. One possible explanation for why this reaction is catalyzed by this trinucleotide (rather than the complementary tetramer) is that the UUU-AAA pairing has the lowest stability and highest flexibility among the 64 trinucleotide conformations, which enables it to bind to Mn²⁺. However, metal ions are not essential for phosphoryl transfer. Some examples of metal-independent catalysis are pancreatic ribonuclease A and hepatitis delta virus (HDV) ribozymes, which use acid-base catalysis to cleave the RNA backbone. Another catalytic activity of ribozymes is the formation of a peptide bond between two amino acids. Ribozymes can facilitate this reaction by reducing the activation entropy.

Activities of Ribozymes

Despite their low abundance in most cells, ribozymes have some vital functions for life. An example of a ribozyme that can catalyze peptide bond formation is the ribosome, the biological machine that translates RNA into proteins. The ribosome is mainly composed of RNA tertiary structural motifs that coordinate metal ions such as Mg²⁺ as cofactors. A model system that mimics this activity is a five-nucleotide RNA that can trans-phenylalanylate a four-nucleotide substrate with three base pairs complementary to the catalyst. This ribozyme is derived from the C3 ribozyme, a self-aminoacylating RNA1. Unlike the C3 ribozyme, the five-nucleotide ribozyme can act in trans and does not require divalent cations for catalysis. The most extensively investigated ribozymes are those that catalyze their own or other RNAs' cleavage, as in the initial discovery. Furthermore, RNA may have a role in catalyzing the folding of prions, which are proteins that adopt pathological conformations. This role is similar to that of chaperonins, which are protein complexes that assist in protein folding.

Artificial Ribozymes

The discovery of ribozymes, RNA molecules that can catalyze chemical reactions, in living organisms has stimulated the research on new synthetic ribozymes created in the laboratory. For instance, some artificial self-cleaving RNAs with high enzymatic activity have been successfully generated. Self-cleaving RNAs have been successfully isolated by in vitro selection of RNAs originating from random-sequence RNAs. The synthetic ribozymes that have been created exhibit diverse structures, ranging from novel ones to those resembling the natural hammerhead ribozyme. More recently, a tethered ribosome has been engineered to work nearly as well as the authentic cellular component that produces all the proteins and enzymes within the cell. This artificial ribosome is called Ribosome-T, or Ribo-T. Artificial ribozymes are generated by using directed evolution techniques. This method exploits the unique property of RNA as both a catalyst and an informational polymer, which allows the researcher to synthesize large populations of RNA catalysts with polymerase enzymes. The ribozymes undergo mutation by being reverse transcribed into various cDNA with reverse transcriptase and amplified with error-prone PCR. The selection criteria in these experiments vary depending on the desired activity. For instance, a ligase ribozyme can be selected by using biotin tags that are covalently attached to the substrate. The active molecules can then be recovered by using a streptavidin matrix that binds to the biotin tags. Furthermore, ribozyme ligases capable of self-replication in about an hour have been developed by using in vitro evolution. They function via the joining of pre-synthesized highly complementary oligonucleotides.

Another active area of research is the creation of artificial self-cleaving riboswitches, also known as aptazymes, which are not true catalysts. Riboswitches are regulatory RNA motifs that alter their structure upon binding to a small molecule ligand and modulate translation. Many natural riboswitches have been identified that recognize various metabolites and other small organic molecules, but only one ribozyme based on a riboswitch has been reported: glmS. The initial work on characterizing self-cleaving riboswitches used theophylline as the ligand. In these studies, an RNA hairpin blocks the ribosome binding site and prevents translation. The regulatory RNA region is cleaved off in the presence of the ligand, such as theophylline, and allows the ribosome to bind and translate the target gene. This RNA engineering work relied on rational design and known RNA structures rather than directed evolution as in the previous examples. More recent work has expanded the ligands used in ribozyme riboswitches to include thymine pyrophosphate. Fluorescence-activated cell sorting has also been employed to engineer aptazymes.

Applications and Challenges of Ribozymes

As mentioned earlier, ribozymes are RNA molecules, and hence their sequences can be easily inserted into any gene, and ribozyme-including RNA transcripts can be produced. The genetic information of every organism is stored in genes, which are transferred to mRNAs before translation into proteins. Thus, the integration of ribozymes into different biological systems enables the control of cellular processes in a variety of ways. Moreover, ribozymes can be applied to various biological systems, such as bacterial, endomembrane, immunological, and plant systems. One area of ribozyme gene therapy is the inhibition of RNA-based viruses. A synthetic ribozyme that targets HIV RNA, called gene shears, has been developed and tested clinically for HIV infection. Similarly, ribozymes have been designed to target the hepatitis C virus RNA, SARS-CoV, adenovirus, and influenza A and B virus RNA. The ribozyme can cleave the conserved regions of the viral genome, which reduces the viral load in mammalian cell culture.

Figure 3. Catalytic RNA, ribozyme, and its applications in synthetic biology. (Park SV. 2019)

Ribozymes have been suggested and developed for the treatment of diseases through gene therapy. A major challenge of using RNA-based enzymes as therapeutics is the short half-life of the catalytic RNA molecules in the body. To overcome this, the 2' position on the ribose is modified to enhance RNA stability.

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