Editing Ribozyme

{{short description|Type of RNA molecules}}
{{About|the chemical|the rock band|Ribozyme (band)}}
[[File:Full length hammerhead ribozyme.png|thumb|3D structure of a [[hammerhead ribozyme]]]]

'''Ribozymes''' ('''ribo'''nucleic acid en'''zyme'''s) are [[RNA]] molecules that have the ability to catalyze specific biochemical reactions, including [[RNA splicing]] in [[gene expression]], similar to the action of protein [[enzymes]]. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like [[DNA]]) and a biological [[catalysis|catalyst]] (like protein enzymes), and contributed to the [[RNA world hypothesis]], which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems.<ref name="pmid6297745"/>

The most common activities of natural or [[Directed evolution|''in vitro'' evolved]] ribozymes are the cleavage (or ligation) of RNA and DNA and peptide bond formation.<ref name="Fedor_2005">{{cite journal | vauthors = Fedor MJ, Williamson JR | title = The catalytic diversity of RNAs | journal = Nature Reviews. Molecular Cell Biology | volume = 6 | issue = 5 | pages = 399–412 | date = May 2005 | pmid = 15956979 | doi = 10.1038/nrm1647 | s2cid = 33304782 }}</ref> For example, the smallest ribozyme known (GUGGC-3') can aminoacylate a GCCU-3' sequence in the presence of PheAMP.<ref>{{cite journal | vauthors = Yarus M | title = The meaning of a minuscule ribozyme | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 366 | issue = 1580 | pages = 2902–2909 | date = October 2011 | pmid = 21930581 | pmc = 3158920 | doi = 10.1098/rstb.2011.0139 }}</ref> Within the [[ribosome]], ribozymes function as part of the large subunit ribosomal RNA to link amino acids during [[Translation (biology)|protein synthesis]]. They also participate in a variety of [[RNA processing]] reactions, including [[RNA splicing]], [[viral replication]], and [[transfer RNA]] biosynthesis. Examples of ribozymes include the [[hammerhead ribozyme]], the [[VS ribozyme]], [[leadzyme]], and the [[hairpin ribozyme]].

Researchers who are investigating the [[origins of life]] through the RNA world hypothesis have been working on discovering a ribozyme with the capacity to self-replicate, which would require it to have the ability to catalytically synthesize polymers of RNA. This should be able to happen in prebiotically plausible conditions with high rates of copying accuracy to prevent degradation of information but also allowing for the occurrence of occasional errors during the copying process to allow for [[History of evolutionary thought|Darwinian evolution]] to proceed.<ref name="Martin_2015">{{cite journal | vauthors = Martin LL, Unrau PJ, Müller UF | title = RNA synthesis by in vitro selected ribozymes for recreating an RNA world | journal = Life | location = Basel, Switzerland | volume = 5 | issue = 1 | pages = 247–68 | date = January 2015 | pmid = 25610978 | pmc = 4390851 | doi = 10.3390/life5010247 | bibcode = 2015Life....5..247M | doi-access = free }}</ref>

Attempts have been made to develop ribozymes as therapeutic agents, as enzymes which target defined RNA sequences for cleavage, as [[biosensor]]s, and for applications in [[functional genomics]] and gene discovery.<ref name="Hean">{{cite book |vauthors=Hean J, Weinberg MS | editor = Morris KL | title = RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity | publisher = Caister Academic Press | location = Norfolk, England | year = 2008 | isbn = 978-1-904455-25-7 | chapter=The Hammerhead Ribozyme Revisited: New Biological Insights for the Development of Therapeutic Agents and for Reverse Genomics Applications | chapter-url = http://www.horizonpress.com/rnareg}}</ref>

==Discovery==
[[File:Ribozyme.jpg|thumb|right|300px|Schematic showing ribozyme cleavage of RNA]]
Before the discovery of ribozymes, [[enzyme]]s—which were defined [solely] as catalytic [[protein]]s—were the only known biological [[catalysis|catalysts]]. In 1967, [[Carl Woese]], [[Francis Crick]], and [[Leslie Orgel]] were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex [[secondary structure]]s.<ref>{{cite book | vauthors = Woese C | title = The Genetic Code | location = New York | publisher = Harper and Row | date = 1967 }}</ref> These ribozymes were found in the [[intron]] of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-[[tRNA]]s. In 1989, [[Thomas R. Cech]] and [[Sidney Altman]] shared the [[Nobel Prize]] in [[chemistry]] for their "discovery of catalytic properties of RNA".<ref>[http://nobelprize.org/chemistry/laureates/1989/ The Nobel Prize in Chemistry 1989] was awarded to [[Thomas R. Cech]] and [[Sidney Altman]] "for their discovery of catalytic properties of RNA".</ref> The term ''ribozyme'' was first introduced by Kelly Kruger ''et al.'' in a paper published in ''[[Cell (journal)|Cell]]'' in 1982.<ref name="pmid6297745">{{cite journal | vauthors = Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR | title = Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena | journal = Cell | volume = 31 | issue = 1 | pages = 147–157 | date = November 1982 | pmid = 6297745 | doi = 10.1016/0092-8674(82)90414-7 | s2cid = 14787080 }}</ref>

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.<ref name="pmid6205343">{{cite journal | vauthors = Visser CM | title = Evolution of biocatalysis 1. Possible pre-genetic-code RNA catalysts which are their own replicase | journal = Origins of Life | volume = 14 | issue = 1–4 | pages = 291–300 | year = 1984 | pmid = 6205343 | doi = 10.1007/BF00933670 | s2cid = 31409366 | bibcode = 1984OrLi...14..291V }}</ref>

In the 1980s, Thomas Cech, at the [[University of Colorado Boulder]], was studying the excision of [[introns]] in a ribosomal RNA gene in ''[[Tetrahymena]] thermophila''. While trying to purify the enzyme responsible for the splicing reaction, he found that the intron could be spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform [[phosphodiester]] bonds. At about the same time, Sidney Altman, a professor at [[Yale University]], was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called [[Ribonuclease P|RNase-P]], which is responsible for conversion of a precursor [[tRNA]] into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year{{Which|date=January 2023}}, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component.

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 chain|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.<ref name="Fedor_2005"/>{{clarify|reason= **This is example is not clear can someone please translate to layman's terms?**"For example, self cleaving ribozyme RNAs and RNase A proteins both cleave phosphate backbones. However, the mechanisms differs. While the RNase A protein uses a coordinating histidine and lysine to attack the backbone, self cleaving ribozyme RNAs carry out an in-line SN2 reaction 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."|date=April 2018}}

Like many protein enzymes, metal binding is also critical to the function of many ribozymes.<ref>{{cite journal | vauthors = Pyle AM | title = Ribozymes: a distinct class of metalloenzymes | journal = Science | volume = 261 | issue = 5122 | pages = 709–714 | date = August 1993 | pmid = 7688142 | doi = 10.1126/science.7688142 | bibcode = 1993Sci...261..709P }}</ref> Often these interactions use both the phosphate backbone and the base of the nucleotide, causing drastic conformational changes.<ref>{{cite journal | vauthors = Freisinger E, Sigel RK | year = 2007 | title = From nucleotides to ribozymes—A comparison of their metal ion binding properties | url = https://www.zora.uzh.ch/id/eprint/59908/1/ZORA_.59908_FromNucleotidesToRibozymes.pdf| journal = Coord. Chem. Rev. | volume = 251 | issue = 13–14| pages = 1834–1851 | doi=10.1016/j.ccr.2007.03.008}}</ref> 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 SN<sub>2</sub> mechanism. Metal ions promote this reaction by first coordinating the phosphate oxygen and later stabling the oxyanion. The second mechanism also follows a SN<sub>2</sub> 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 Mn<sup>2+</sup>. 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 Mn<sup>2+</sup>.<ref name=":0">{{cite journal | vauthors = Pyle AM | title = Ribozymes: a distinct class of metalloenzymes | journal = Science | volume = 261 | issue = 5122 | pages = 709–714 | date = August 1993 | pmid = 7688142 | doi = 10.1126/science.7688142 | bibcode = 1993Sci...261..709P | jstor = 2882234 }}</ref>

Phosphoryl transfer can also be catalyzed without metal ions. For example, pancreatic ribonuclease A and [[hepatitis delta virus]] (HDV) ribozymes can catalyze the cleavage of RNA backbone through acid-base catalysis without metal ions.<ref name=":2">{{cite journal | vauthors = Lilley DM | title = Mechanisms of RNA catalysis | journal = Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences | volume = 366 | issue = 1580 | pages = 2910–2917 | date = October 2011 | pmid = 21930582 | pmc = 3158914 | doi = 10.1098/rstb.2011.0132 | jstor = 23035661 }}</ref><ref name=":1">{{cite journal | vauthors = Doudna JA, Cech TR | title = The chemical repertoire of natural ribozymes | journal = Nature | volume = 418 | issue = 6894 | pages = 222–228 | date = July 2002 | pmid = 12110898 | doi = 10.1038/418222a | s2cid = 4417095 | bibcode = 2002Natur.418..222D }}</ref> Hairpin ribozyme can also catalyze the self-cleavage of RNA without metal ions, but the mechanism for this is still unclear.<ref name=":1" />

Ribozyme can also catalyze the formation of peptide bond between adjacent amino acids by lowering the activation entropy.<ref name=":2" />

[[File:Ribozyme structure picutres.png|thumb|center|550px|alt=Ribozyme structure pictures|Image showing the diversity of ribozyme structures. From left to right: leadzyme, hammerhead ribozyme, twister ribozyme]]

==Activities==
[[Image:Protein translation.gif|thumb|300px| A [[ribosome]] is a [[biological machine]] that utilizes a ribozyme to [[translation (biology)|translate]] RNA into proteins.]]
Although ribozymes are quite rare in most cells, their roles are sometimes essential to life. For example, the functional part of the [[ribosome]], the [[biological machine]] that [[translation (biology)|translates]] RNA into proteins, is fundamentally a ribozyme, composed of [[RNA Tertiary Structure|RNA tertiary structural motifs]] that are often coordinated to metal ions such as [[Mg2+|Mg]]<sup>2+</sup> as [[cofactor (biochemistry)|cofactor]]s.<ref>{{cite journal | vauthors = Ban N, Nissen P, Hansen J, Moore PB, Steitz TA | title = The complete atomic structure of the large ribosomal subunit at 2.4 A resolution | journal = Science | volume = 289 | issue = 5481 | pages = 905–920 | date = August 2000 | pmid = 10937989 | doi = 10.1126/science.289.5481.905 | citeseerx = 10.1.1.58.2271 | bibcode = 2000Sci...289..905B }}</ref> In a model system, there is no requirement for [[divalent]] [[cations]] in a five-nucleotide RNA catalyzing ''trans''-[[Phenylalanine|phenylalanation]] of a four-nucleotide substrate with 3 base pairs complementary with the catalyst, where the catalyst/substrate were devised by truncation of the C3 ribozyme.<ref name="pmid20176971">{{cite journal | vauthors = Turk RM, Chumachenko NV, Yarus M | title = Multiple translational products from a five-nucleotide ribozyme | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 107 | issue = 10 | pages = 4585–4589 | date = March 2010 | pmid = 20176971 | pmc = 2826339 | doi = 10.1073/pnas.0912895107 | doi-access = free | bibcode = 2010PNAS..107.4585T }}</ref>

The best-studied ribozymes are probably those that cut themselves or other RNAs, as in the original discovery by Cech<ref>{{cite journal | vauthors = Cech TR | title = Structural biology. The ribosome is a ribozyme | journal = Science | volume = 289 | issue = 5481 | pages = 878–879 | date = August 2000 | pmid = 10960319 | doi = 10.1126/science.289.5481.878 | s2cid = 24172338 }}</ref> and Altman.<ref>{{cite journal | vauthors = Altman S | title = Nobel lecture. Enzymatic cleavage of RNA by RNA | journal = Bioscience Reports | volume = 10 | issue = 4 | pages = 317–337 | date = August 1990 | pmid = 1701103 | doi = 10.1007/BF01117232 | s2cid = 12733970 }}</ref> 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.<ref>{{cite journal | vauthors = Walter NG, Engelke DR | title = Ribozymes: catalytic RNAs that cut things, make things, and do odd and useful jobs | journal = Biologist | volume = 49 | issue = 5 | pages = 199–203 | date = October 2002 | pmid = 12391409 | pmc = 3770912 }}</ref>

RNA may catalyze [[protein folding|folding]] of the pathological [[Chemical conformation|protein conformation]] of a [[prion]] in a manner similar to that of a [[Chaperone (protein)|chaperonin]].<ref name="pmid15014886">{{cite journal | vauthors = Supattapone S | title = Prion protein conversion in vitro | journal = Journal of Molecular Medicine | volume = 82 | issue = 6 | pages = 348–356 | date = June 2004 | pmid = 15014886 | doi = 10.1007/s00109-004-0534-3 | s2cid = 24908667 }}</ref>

== Ribozymes and the origin of life ==
RNA can also act as a hereditary molecule, which encouraged [[Walter Gilbert]] to propose that in the distant past, the [[cell (biology)|cell]] used RNA as both the genetic material and the structural and catalytic molecule rather than dividing these functions between [[DNA]] and [[protein]] as they are today; this hypothesis is known as the "[[RNA world hypothesis]]" of the [[origin of life]].<ref>{{cite journal | vauthors = Gilbert W | title = Origin of life: The RNA world | journal = Nature | volume = 319 | issue = 6055 | pages = 618 |date= 1986 | doi = 10.1038/319618a0 | bibcode = 1986Natur.319..618G | s2cid = 8026658 | doi-access = free }}</ref> Since [[nucleotide]]s and [[RNA]] (and thus ribozymes) can arise by inorganic chemicals, they are candidates for the first [[enzyme]]s, and in fact, the first "replicators" (i.e., information-containing macro-molecules that replicate themselves). An example of a self-replicating ribozyme that ligates two substrates to generate an exact copy of itself was described in 2002.<ref>{{cite journal | vauthors = Paul N, Joyce GF | title = A self-replicating ligase ribozyme | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 20 | pages = 12733–12740 | date = October 2002 | pmid = 12239349 | pmc = 130529 | doi = 10.1073/pnas.202471099 | doi-access = free | bibcode = 2002PNAS...9912733P }}</ref>
The discovery of the catalytic activity of RNA solved the "chicken and egg" paradox of the origin of life, solving the problem of origin of peptide and nucleic acid central dogma. According to this scenario, at the origin of life, all enzymatic activity and genetic information encoding was done by one molecule: RNA.

Ribozymes have been produced in the [[laboratory]] that are capable of catalyzing the synthesis of other RNA molecules from activated [[monomer]]s under very specific conditions, these molecules being known as [[RNA polymerase]] ribozymes.<ref name="pmid11358999">{{cite journal | vauthors = Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP | title = RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension | journal = Science | volume = 292 | issue = 5520 | pages = 1319–1325 | date = May 2001 | pmid = 11358999 | doi = 10.1126/science.1060786 | s2cid = 14174984 | citeseerx = 10.1.1.70.5439 | bibcode = 2001Sci...292.1319J }}</ref> The first RNA polymerase ribozyme was reported in 1996, and was capable of synthesizing RNA polymers up to 6 nucleotides in length.<ref name="Ekland_1996">{{cite journal | vauthors = Ekland EH, Bartel DP | title = RNA-catalysed RNA polymerization using nucleoside triphosphates | journal = Nature | volume = 382 | issue = 6589 | pages = 373–6 | date = July 1996 | pmid = 8684470 | doi = 10.1038/382373a0 | bibcode = 1996Natur.382..373E | s2cid = 4367137 }}</ref> [[Mutagenesis]] and selection has been performed on an RNA ligase ribozyme from a large pool of random RNA sequences,<ref name="Bartel_1993">{{cite journal | vauthors = Bartel DP, Szostak JW | title = Isolation of new ribozymes from a large pool of random sequences [see comment] | journal = Science | location = New York, N.Y. | volume = 261 | issue = 5127 | pages = 1411–8 | date = September 1993 | pmid = 7690155 | doi = 10.1126/science.7690155 | url = }}</ref> resulting in isolation of the improved "Round-18" polymerase ribozyme in 2001 which could catalyze RNA polymers now up to 14 nucleotides in length.<ref name="Johnston_2001">{{cite journal | vauthors = Johnston WK, Unrau PJ, Lawrence MS, Glasner ME, Bartel DP | title = RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension | journal = Science | location = New York, N.Y. | volume = 292 | issue = 5520 | pages = 1319–25 | date = May 2001 | pmid = 11358999 | doi = 10.1126/science.1060786 | bibcode = 2001Sci...292.1319J | s2cid = 14174984 }}</ref> Upon application of further selection on the Round-18 ribozyme, the B6.61 ribozyme was generated and was able to add up to 20 [[nucleotides]] to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds.<ref name="pmid17586759">{{cite journal | vauthors = Zaher HS, Unrau PJ | title = Selection of an improved RNA polymerase ribozyme with superior extension and fidelity | journal = RNA | volume = 13 | issue = 7 | pages = 1017–1026 | date = July 2007 | pmid = 17586759 | pmc = 1894930 | doi = 10.1261/rna.548807 }}</ref>

The rate at which ribozymes can polymerize an RNA sequence multiples substantially when it takes place within a [[micelle]].<ref name="Müller_2008">{{cite journal | vauthors = Müller UF, Bartel DP | title = Improved polymerase ribozyme efficiency on hydrophobic assemblies | journal = RNA | location = New York, N.Y. | volume = 14 | issue = 3 | pages = 552–62 | date = March 2008 | pmid = 18230767 | pmc = 2248263 | doi = 10.1261/rna.494508 }}</ref>

The next ribozyme discovered was the "tC19Z" ribozyme, which can add up to 95 [[nucleotides]] with a fidelity of 0.0083 mutations/nucleotide.<ref name="pmid21474753">{{cite journal | vauthors = Wochner A, Attwater J, Coulson A, Holliger P | title = Ribozyme-catalyzed transcription of an active ribozyme | journal = Science | volume = 332 | issue = 6026 | pages = 209–212 | date = April 2011 | pmid = 21474753 | doi = 10.1126/science.1200752 | s2cid = 39990861 | bibcode = 2011Sci...332..209W }}</ref> Next, the "tC9Y" ribozyme was discovered by researchers and was further able to synthesize RNA strands up to 206 nucleotides long in the eutectic phase conditions at below-zero temperature,<ref name="Attwater_2013">{{cite journal | vauthors = Attwater J, Wochner A, Holliger P | title = In-ice evolution of RNA polymerase ribozyme activity | journal = Nature Chemistry | volume = 5 | issue = 12 | pages = 1011–8 | date = December 2013 | pmid = 24256864 | pmc = 3920166 | doi = 10.1038/nchem.1781 | bibcode = 2013NatCh...5.1011A }}</ref> conditions previously shown to promote ribozyme polymerase activity.<ref name="Attwater_2010">{{cite journal | vauthors = Attwater J, Wochner A, Pinheiro VB, Coulson A, Holliger P | title = Ice as a protocellular medium for RNA replication | journal = Nature Communications | volume = 1 | issue = 6| pages = 76 | date = September 2010 | pmid = 20865803 | doi = 10.1038/ncomms1076 | bibcode = 2010NatCo...1...76A | doi-access = free }}</ref>

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.<ref name="Tagami_2017">{{cite journal | vauthors = Tagami S, Attwater J, Holliger P | title = Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function | journal = Nature Chemistry | volume = 9 | issue = 4 | pages = 325–332 | date = April 2017 | pmid = 28338682 | pmc = 5458135 | doi = 10.1038/nchem.2739 | bibcode = 2017NatCh...9..325T }}</ref>

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.<ref name="Horning_2016">{{cite journal | vauthors = Horning DP, Joyce GF | title = Amplification of RNA by an RNA polymerase ribozyme | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 35 | pages = 9786–91 | date = August 2016 | pmid = 27528667 | pmc = 5024611 | doi = 10.1073/pnas.1610103113 | bibcode = 2016PNAS..113.9786H | doi-access = free }}</ref> Starting with the 24-3 ribozyme, Tjhung et al.<ref name="Tjhung_2020">{{cite journal | vauthors = Tjhung KF, Shokhirev MN, Horning DP, Joyce GF | title = An RNA polymerase ribozyme that synthesizes its own ancestor | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 117 | issue = 6 | pages = 2906–2913 | date = February 2020 | pmid = 31988127 | pmc = 7022166 | doi = 10.1073/pnas.1914282117 | bibcode = 2020PNAS..117.2906T | doi-access = free }}</ref> applied another fourteen rounds of selection to obtain an RNA polymerase ribozyme by [[directed evolution|''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.<ref>{{cite journal | vauthors = Portillo X, Huang YT, Breaker RR, Horning DP, Joyce GF | date = 2021 | title = Witnessing the structural evolution of an RNA enzyme. | journal = eLife | volume = 10 | pages = e71557 | doi = 10.7554/eLife.71557 | pmid = 34498588 | pmc = 8460264 | doi-access = free }}</ref>

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.<ref name="Attwater_2018">{{cite journal | vauthors = Attwater J, Raguram A, Morgunov AS, Gianni E, Holliger P | title = Ribozyme-catalysed RNA synthesis using triplet building blocks | journal = eLife | volume = 7 | issue = | pages = | date = May 2018 | pmid = 29759114 | pmc = 6003772 | doi = 10.7554/eLife.35255 | doi-access = free }}</ref>

A highly evolved{{Vague|reason=What does "highly evolved" mean? Strange phrasing.|date={{CURRENTMONTHNAME}} {{CURRENTYEAR}}}} RNA polymerase ribozyme was able to function as a [[reverse transcriptase]], that is, it can synthesize a DNA copy using an RNA template.<ref name="Samanta_2017">{{cite journal | vauthors = Samanta B, Joyce GF | title = A reverse transcriptase ribozyme | journal = eLife | volume = 6 | issue = | pages = | date = September 2017 | pmid = 28949294 | pmc = 5665644 | doi = 10.7554/eLife.31153 | doi-access = free }}</ref> Such an activity is considered{{By whom|date=April 2022}} 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.<ref name="pmid33737482">{{cite journal | vauthors = Cojocaru R, Unrau PJ | title = Processive RNA polymerization and promoter recognition in an RNA World | journal = Science | volume = 371 | issue = 6535 | pages = 1225–1232 | date = March 2021 | pmid = 33737482 | doi = 10.1126/science.abd9191 | bibcode = 2021Sci...371.1225C | s2cid = 232271298 }}</ref>

==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<ref name="TangBreaker">{{cite journal | vauthors = Tang J, Breaker RR | title = Structural diversity of self-cleaving ribozymes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 11 | pages = 5784–5789 | date = May 2000 | pmid = 10823936 | pmc = 18511 | doi = 10.1073/pnas.97.11.5784 | doi-access = free | bibcode = 2000PNAS...97.5784T }}</ref> 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 [[Northwestern University]] and the [[University of Illinois Chicago]] engineered a tethered ribosome that works nearly as well as the authentic cellular component that produces all the proteins and enzymes within the cell. Called [[Ribosome-T]], or Ribo-T, the artificial ribosome was created by Michael Jewett and Alexander Mankin.<ref>[http://www.northwestern.edu/newscenter/stories/2015/07/artificial-ribosomes.html#sthash.CiTI1pw3.dpuf Engineer and Biologist Design First Artificial Ribosome - Designer ribosome could lead to new drugs and next-generation biomaterials] published on July 31, 2015 by Northwestern University</ref> The techniques used to create artificial ribozymes involve directed evolution. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using [[polymerase]] enzymes. The ribozymes are mutated by reverse transcribing them with [[reverse transcriptase]] into various [[cDNA]] and amplified with [[error-prone PCR]]. The selection parameters in these experiments often differ. One approach for selecting a [[ligase ribozyme]] involves using [[biotin]] tags, which are [[Covalent bond|covalently]] linked to the substrate. If a molecule possesses the desired [[ligase]] activity, a [[streptavidin]] matrix can be used to recover the active molecules.

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.<ref name="pmid19131595">{{cite journal | vauthors = Lincoln TA, Joyce GF | title = Self-sustained replication of an RNA enzyme | journal = Science | volume = 323 | issue = 5918 | pages = 1229–1232 | date = February 2009 | pmid = 19131595 | pmc = 2652413 | doi = 10.1126/science.1167856 | bibcode = 2009Sci...323.1229L }}</ref>

Although not true catalysts, the creation of artificial self-cleaving [[Riboswitch|riboswitches]], termed ''aptazymes'', has also been an active area of research. Riboswitches are regulatory RNA motifs that change their structure in response to a small molecule ligand to regulate translation. While there are many known natural riboswitches that bind a wide array of metabolites and other small organic molecules, only one ribozyme based on a riboswitch has been described: [[GlmS glucosamine-6-phosphate activated ribozyme|''glmS'']].<ref>{{cite journal | vauthors = Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR | title = Control of gene expression by a natural metabolite-responsive ribozyme | journal = Nature | date = March 2004 | volume = 428 | issue = 6980 | pages = 281–286 | doi = 10.1038/nature02362 | pmid = 15029187 | bibcode = 2004Natur.428..281W | s2cid = 4301164 }}</ref> Early work in characterizing self-cleaving riboswitches was focused on using [[theophylline]] as the ligand. In these studies, an RNA hairpin is formed which blocks the [[ribosome binding site]], thus inhibiting translation. In the presence of the [[ligand]], in these cases theophylline, the regulatory RNA region is cleaved off, allowing the ribosome to bind and translate the target gene. Much of this RNA engineering work was based on rational design and previously determined RNA structures rather than directed evolution as in the above examples. More recent work has broadened the ligands used in ribozyme riboswitches to include thymine pyrophosphate. [[Fluorescence-activated cell sorting]] has also been used to engineering aptazymes.<ref>{{cite journal | vauthors = Lynch SA, Gallivan JP | title = A flow cytometry-based screen for synthetic riboswitches | journal = Nucleic Acids Research | volume = 37 | issue = 1 | pages = 184–192 | date = January 2009 | pmid = 19033367 | pmc = 2615613 | doi = 10.1093/nar/gkn924 }}</ref>

== Applications ==<!-- gene shears links here -->

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.<ref name="pmid11249628">{{cite journal | vauthors = de Feyter R, Li P | title = Technology evaluation: HIV ribozyme gene therapy, Gene Shears Pty Ltd | journal = Current Opinion in Molecular Therapeutics | volume = 2 | issue = 3 | pages = 332–335 | date = June 2000 | pmid = 11249628 }}</ref><ref name="pmid16426595">{{cite journal | vauthors = Khan AU | title = Ribozyme: a clinical tool | journal = Clinica Chimica Acta; International Journal of Clinical Chemistry | volume = 367 | issue = 1–2 | pages = 20–27 | date = May 2006 | pmid = 16426595 | doi = 10.1016/j.cca.2005.11.023 }}</ref>

Similarly, ribozymes have been designed to target the [[hepatitis C virus]] RNA, [[SARS coronavirus]] (SARS-CoV),<ref name=":3">{{cite journal | vauthors = Asha K, Kumar P, Sanicas M, Meseko CA, Khanna M, Kumar B | title = Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections | journal = Journal of Clinical Medicine | volume = 8 | issue = 1 | pages = 6 | date = December 2018 | pmid = 30577479 | pmc = 6351902 | doi = 10.3390/jcm8010006 | doi-access = free }}</ref> [[Adenovirus]]<ref name=":3" /> and [[Influenza A virus|influenza A]] and [[Influenza B virus|B]] virus RNA.<ref>{{cite journal | vauthors = Khanna M, Saxena L, Rajput R, Kumar B, Prasad R | title = Gene silencing: a therapeutic approach to combat influenza virus infections | journal = Future Microbiology | volume = 10 | issue = 1 | pages = 131–140 | date = 2015 | pmid = 25598342 | doi = 10.2217/fmb.14.94 }}</ref><ref>{{cite journal | vauthors = Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC | title = Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site | journal = Molecular Biotechnology | volume = 51 | issue = 1 | pages = 27–36 | date = May 2012 | pmid = 21744034 | doi = 10.1007/s12033-011-9437-z | s2cid = 45686564 }}</ref><ref>{{cite journal | vauthors = Kumar B, Asha K, Khanna M, Ronsard L, Meseko CA, Sanicas M | title = The emerging influenza virus threat: status and new prospects for its therapy and control | journal = Archives of Virology | volume = 163 | issue = 4 | pages = 831–844 | date = April 2018 | pmid = 29322273 | pmc = 7087104 | doi = 10.1007/s00705-018-3708-y }}</ref><ref name=":3" /> The ribozyme is able to cleave the conserved regions of the virus's genome, which has been shown to reduce the virus in mammalian cell culture.<ref>{{cite journal | vauthors = Lieber A, He CY, Polyak SJ, Gretch DR, Barr D, Kay MA | title = Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes | journal = Journal of Virology | volume = 70 | issue = 12 | pages = 8782–8791 | date = December 1996 | pmid = 8971007 | pmc = 190975 | doi = 10.1128/JVI.70.12.8782-8791.1996 }}</ref> Despite these efforts by researchers, these projects have remained in the preclinical stage.

==Known ribozymes==
Well-validated naturally occurring ribozyme classes:
{{div col|colwidth=30em}}
* [[GIR1 branching ribozyme]]<ref name="pmid16141078">{{cite journal | vauthors = Nielsen H, Westhof E, Johansen S | title = An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme | journal = Science | volume = 309 | issue = 5740 | pages = 1584–1587 | date = September 2005 | pmid = 16141078 | doi = 10.1126/science.1113645 | s2cid = 37002071 | bibcode = 2005Sci...309.1584N }}</ref>
* [[GlmS glucosamine-6-phosphate activated ribozyme|''glmS'' ribozyme]]
* [[Group I catalytic intron|Group I]] self-splicing intron
* [[Group II intron|Group II]] self-splicing intron – [[Spliceosome]] is likely derived from Group II self-splicing ribozymes.<ref name="pmid24196718">{{cite journal | vauthors = Fica SM, Tuttle N, Novak T, Li NS, Lu J, Koodathingal P, Dai Q, Staley JP, Piccirilli JA | display-authors = 6 | title = RNA catalyses nuclear pre-mRNA splicing | journal = Nature | volume = 503 | issue = 7475 | pages = 229–234 | date = November 2013 | pmid = 24196718 | pmc = 4666680 | doi = 10.1038/nature12734 | bibcode = 2013Natur.503..229F }}</ref>
* [[Hairpin ribozyme]]
* [[Hammerhead ribozyme]]
* [[Hepatitis delta virus ribozyme|HDV ribozyme]]
* [[ribosomal RNA|rRNA]] – Found in all living cells and links [[amino acids]] to form [[protein]]s.
* [[RNase P]]
* [[Twister ribozyme]]
* [[Twister sister ribozyme]]
* [[VS ribozyme]]
* [[Pistol ribozyme]]
* [[Hatchet ribozyme]]
* [[Viroids]]{{div col end}}

== See also ==
{{div col|colwidth=30em}}
*[[Deoxyribozyme]]
*[[Spiegelman Monster]]
*[[Catalysis]]
*[[Enzyme]]
*[[RNA world hypothesis]]
*[[Peptide nucleic acid]]
*[[Nucleic acid analogues]]
*[[PAH world hypothesis]]
*[[Systematic Evolution of Ligands by Exponential Enrichment|SELEX]]
*[[OLE RNA]]
{{div col end}}

==Notes and references==
{{reflist}}

== Further reading ==
{{refbegin|33em}}
* {{cite book | vauthors = Sigel A, Sigel H, Sigel RK | editor-first1 = Astrid | editor-first2 = Helmut | editor-first3 = Roland K O | editor-first4 = Astrid | editor-first5 = Helmut | editor-first6 = Roland K O | editor-first7 = Astrid | editor-first8 = Helmut | editor-first9 = Roland K O | editor-first10 = Astrid | editor-first11 = Helmut | editor-first12 = Roland K O | editor-last1 = Sigel | editor-last2 = Sigel | editor-last3 = Sigel | editor-last4 = Sigel | editor-last5 = Sigel | editor-last6 = Sigel | editor-last7 = Sigel | editor-last8 = Sigel | editor-last9 = Sigel | editor-last10 = Sigel | editor-last11 = Sigel | editor-last12 = Sigel | title = Structural and catalytic roles of metal ions in RNA | journal = Metal Ions in Life Sciences | volume = 9 | pages = vii–ix | year = 2011 | pmid = 22010266 | doi = 10.1039/9781849732512 | publisher = RSC Publishing | isbn = 978-1-84973-251-2 }}
* {{cite book | vauthors = Johnson-Buck AE, McDowell SE, Walter NG | title = Structural and Catalytic Roles of Metal Ions in RNA | chapter = Metal ions: supporting actors in the playbook of small ribozymes | journal = Metal Ions in Life Sciences | volume = 9 | pages = 175–96 | year = 2011 | pmid = 22010272 | pmc = 3365584 | doi = 10.1039/9781849732512-00175 | isbn = 978-1-84973-094-5 }}
* {{cite book | vauthors = Donghi D, Schnabl J | title = Structural and Catalytic Roles of Metal Ions in RNA | chapter = Multiple roles of metal ions in large ribozymes | journal = Metal Ions in Life Sciences | volume = 9 | pages = 197–234 | year = 2011 | pmid = 22010273 | doi = 10.1039/9781849732512-00197 | isbn = 978-1-84973-094-5 }}
* {{cite book | vauthors = Trappl K, Polacek N | title = Structural and Catalytic Roles of Metal Ions in RNA | chapter = The ribosome: a molecular machine powered by RNA | journal = Metal Ions in Life Sciences | volume = 9 | pages = 253–75 | year = 2011 | pmid = 22010275 | doi = 10.1039/9781849732512-00253 | isbn = 978-1-84973-094-5 }}
* {{cite book | vauthors = Suga H, Futai K, Jin K | title = Structural and Catalytic Roles of Metal Ions in RNA | chapter = Metal ion requirements in artificial ribozymes that catalyze aminoacylation and redox reactions | journal = Metal Ions in Life Sciences | volume = 9 | pages = 277–97 | year = 2011 | pmid = 22010276 | doi = 10.1039/9781849732512-00277 | isbn = 978-1-84973-094-5 }}
* {{cite book | vauthors = Wedekind JE | title = Structural and Catalytic Roles of Metal Ions in RNA | chapter = Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective | journal = Metal Ions in Life Sciences | volume = 9 | pages = 299–345 | year = 2011 | pmid = 22010277 | doi = 10.1039/9781849732512-00299 | isbn = 978-1-84973-094-5 }}
* {{cite journal | vauthors = Doherty EA, Doudna JA | title = Ribozyme structures and mechanisms | journal = Annual Review of Biophysics and Biomolecular Structure | volume = 30 | pages = 457–475 | year = 2001 | pmid = 11441810 | doi = 10.1146/annurev.biophys.30.1.457 }}
* {{cite journal | vauthors = Joyce GF | title = Directed evolution of nucleic acid enzymes | journal = Annual Review of Biochemistry | volume = 73 | pages = 791–836 | year = 2004 | pmid = 15189159 | doi = 10.1146/annurev.biochem.73.011303.073717 }}
* {{cite journal | vauthors = Ikawa Y, Tsuda K, Matsumura S, Inoue T | title = De novo synthesis and development of an RNA enzyme | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 101 | issue = 38 | pages = 13750–13755 | date = September 2004 | pmid = 15365187 | pmc = 518828 | doi = 10.1073/pnas.0405886101 | doi-access = free | bibcode = 2004PNAS..10113750I }}
{{refend}}

== External links ==
*[https://www.ibiology.org/genetics-and-gene-regulation/discovering-ribozymes/ Tom Cech's Short Talk: "Discovering Ribozymes"]

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