Ribozyme

Ribozymes (ribonucleic acid enzymes) are

Attempts have been made to develop ribozymes as therapeutic agents, as enzymes which target defined RNA sequences for cleavage, as biosensors, and for applications in functional genomics and gene discovery.^[5]

Discovery

Before the discovery of ribozymes,

It had been a firmly established belief in biology that catalysis was reserved for proteins. However, the idea of RNA catalysis is motivated in part by the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? The concept of "ribonucleic acids as catalysts" circumvents this problem. RNA, in essence, can be both the chicken and the egg.^[9]

In the 1980s, Thomas Cech, at the

Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin – or hammerhead – shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme were made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

Structure and mechanism

Despite having only four choices for each monomer unit (nucleotides), compared to 20 amino acid side chains found in proteins, ribozymes have diverse structures and mechanisms. In many cases they are able to mimic the mechanism used by their protein counterparts. For example, in self cleaving ribozyme RNAs, an in-line SN2 reaction is carried out using the 2’ hydroxyl group as a nucleophile attacking the bridging phosphate and causing 5’ oxygen of the N+1 base to act as a leaving group. In comparison, RNase A, a protein that catalyzes the same reaction, uses a coordinating histidine and lysine to act as a base to attack the phosphate backbone.^{[2][clarification needed]}

Like many protein enzymes, metal binding is also critical to the function of many ribozymes.^[10] Often these interactions use both the phosphate backbone and the base of the nucleotide, causing drastic conformational changes.^[11] There are two mechanism classes for the cleavage of a phosphodiester backbone in the presence of metal. In the first mechanism, the internal 2’- OH group attacks the phosphorus center in a SN₂ mechanism. Metal ions promote this reaction by first coordinating the phosphate oxygen and later stabling the oxyanion. The second mechanism also follows a SN₂ displacement, but the nucleophile comes from water or exogenous hydroxyl groups rather than RNA itself. 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⁺. The reason why this trinucleotide (rather than the complementary tetramer) catalyzes this reaction may be because the UUU-AAA pairing is the weakest and most flexible trinucleotide among the 64 conformations, which provides the binding site for Mn²⁺.^[12]

Phosphoryl transfer can also be catalyzed without metal ions. For example, pancreatic ribonuclease A and

Ribozyme can also catalyze the formation of peptide bond between adjacent amino acids by lowering the activation entropy.[13]

Activities

The best-studied ribozymes are probably those that cut themselves or other RNAs, as in the original discovery by Cech[17] and Altman.^[18] However, ribozymes can be designed to catalyze a range of reactions, many of which may occur in life but have not been discovered in cells.^[19]

RNA may catalyze

The rate at which ribozymes can polymerize an RNA sequence multiples substantially when it takes place within a micelle.^[28]

The next ribozyme discovered was the "tC19Z" ribozyme, which can add up to 95

The RNA polymerase ribozyme (RPR) called tC9-4M was able to polymerize RNA chains longer than itself (i.e. longer than 177 nt) in magnesium ion concentrations close to physiological levels, whereas earlier RPRs required prebiotically implausible concentrations of up to 200 mM. The only factor required for it to achieve this was the presence of a very simple amino acid polymer called lysine decapeptide.[32]

The most complex RPR synthesized by that point was called 24-3, which was newly capable of polymerizing the sequences of a substantial variety of nucleotide sequences and navigating through complex secondary structures of RNA substrates inaccessible to previous ribozymes. In fact, this experiment was the first to use a ribozyme to synthesize a tRNA molecule.^[33] Starting with the 24-3 ribozyme, Tjhung et al.^[34] applied another fourteen rounds of selection to obtain an RNA polymerase ribozyme by in vitro evolution termed '38-6' that has an unprecedented level of activity in copying complex RNA molecules. However, this ribozyme is unable to copy itself and its RNA products have a high mutation rate. In a subsequent study, the researchers began with the 38-6 ribozyme and applied another 14 rounds of selection to generate the '52-2' ribozyme, which compared to 38-6, was again many times more active and could begin generating detectable and functional levels of the class I ligase, although it was still limited in its fidelity and functionality in comparison to copying of the same template by proteins such as the T7 RNA polymerase.^[35]

An RPR called t5(+1) adds triplet nucleotides at a time instead of just one nucleotide at a time. This heterodimeric RPR can navigate secondary structures inaccessible to 24-3, including hairpins. In the initial pool of RNA variants derived only from a previously synthesized RPR known as the Z RPR, two sequences separately emerged and evolved to be mutualistically dependent on each other. The Type 1 RNA evolved to be catalytically inactive, but complexing with the Type 5 RNA boosted its polymerization ability and enabled intermolecular interactions with the RNA template substrate obviating the need to tether the template directly to the RNA sequence of the RPR, which was a limitation of earlier studies. Not only did t5(+1) not need tethering to the template, but a primer was not needed either as t5(+1) had the ability to polymerize a template in both 3' → 5' and 5' 3 → 3' directions.^[36]

A highly evolved^[vague] RNA polymerase ribozyme was able to function as a reverse transcriptase, that is, it can synthesize a DNA copy using an RNA template.^[37] Such an activity is considered^{[by whom?]} to have been crucial for the transition from RNA to DNA genomes during the early history of life on earth. Reverse transcription capability could have arisen as a secondary function of an early RNA-dependent RNA polymerase ribozyme.

An RNA sequence that folds into a ribozyme is capable of invading duplexed RNA, rearranging into an open holopolymerase complex, and then searching for a specific RNA promoter sequence, and upon recognition rearrange again into a processive form that polymerizes a complementary strand of the sequence. This ribozyme is capable of extending duplexed RNA by up to 107 nucleotides, and does so without needing to tether the sequence being polymerized.^[38]

Artificial ribozymes

Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificially produced self-cleaving RNAs with good enzymatic activity have been produced. Tang and Breaker^[39] isolated self-cleaving RNAs by in vitro selection of RNAs originating from random-sequence RNAs. Some of the synthetic ribozymes that were produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme.

In 2015, researchers at

Lincoln and Joyce used in vitro evolution to develop ribozyme ligases capable of self-replication in about an hour, via the joining of pre-synthesized highly complementary oligonucleotides.[41]

Although not true catalysts, the creation of artificial self-cleaving

Ribozymes have been proposed and developed for the treatment of disease through gene therapy. One major challenge of using RNA-based enzymes as a therapeutic is the short half-life of the catalytic RNA molecules in the body. To combat this, the 2’ position on the ribose is modified to improve RNA stability. One area of ribozyme gene therapy has been the inhibition of RNA-based viruses.

A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection.^[44][45]

Similarly, ribozymes have been designed to target the

See also

Notes and references