Slide 1: Good Morning To All! Slide 2: ABZYMES & NON-PROTEIN ENZYMES R.MAREESWARAN, FIRST YEAR M.Sc.

., BIOCHEMISTRY, BHARATHIDASAN UNIVERSITY, TRICHY ABZYMES: ABZYMES Abzyme A nti b ody + En zyme Catmab Catalytic monoclonal antibody In 1986 Peter Schultz & Richard Lerner Presence - usually artificial constructs Normal humans and patients with autoimmune diseases Potential tools in Biotechnology (E.g.) To perform specific action on DNA Similarities between enzymes and antibodies: Similarities between enzymes and antibodies Binds with substrates by weak, non-covalent bonds Exhibits high specificity and high affinity Decrease activation energy and stabilize transition state Difference is, Enzymes modify covalent bonds in substrate and the Ab does not Development of First Abzyme: Monoclonal antibodies MCAb incubate with ester MCAb hydrolyzed ester specifically Catalytic activity - 1000 foldsDevelopment of First Abzyme Hapten-Carrier complex Synthesized Hapten resembles Transition state analogue of an ester Mice immunized with hapten complex Spleen cells fused with myeloma cells Abzyme in treatment of Cocaine addiction: Abzyme in treatment of Cocaine addiction Addictive drugs binds Neurotransmitter receptor Cocaine blocks neurotransmitter reuptake Ab against cocaine hydrolyze cocaine & no prolongation of neural stimulas Abzymes in Tumour treatment: Abzymes in Tumour treatment Tumour cells have specialized antigens on their surface Prodrugs - nontoxic drug became cytotoxic on cleavage by specifically engineered abzymes First abzyme give through blood stream & binds with tumour antigen on the cancer cells surface Prodrug administered secondarily & cleaved by abzyme bind with tumour antigen RIBOZYMES: RIBOZYMES Ribozyme Ribo nucleic acid en zyme RNA enzyme, catalytic RNA - RNA molecule that catalyze chemical reactions Play important role as therapeutic agents, biosensors and for applications in functional genomics and gene discovery Slide 9: A brief History 1982:Self-splicing in Tetrahymena thermophila pre-rRNA (group I intron) Kruger et al, and Thomas Cech , Cell 31, 147-157 (1982) 1983:RNAse P is a ribozyme Guerrier-Takada et al, and Sidney Altman , Cell, 35, 849-857 (1983) Slide 10: Known Ribozymes Hairpin ribozyme (plan virus) Hammerhead ribozyme HDV ribozyme Mammalian CPEB3 ribozyme glmS ribozyme Hepatitis delta ribozyme (human virus) Neurospora VS ribozyme (mitochondrial RNA) Group I and Group II intron ribozyme (rRNA and mt RNA) RNAse P (tRNA maturation) Peptidyl transferase 23S rRNA GIR1 branching ribozyme Ribosome (translation) Spliceosome (splicing) RNase P: RNase P Type of ribonuclease cleaves RNA RNA in nature &acts as Protein enzymes Sidney Altman -1970 Found in E.Coli , yeasts and humans E.Coli contain protein called C5 protein whereas the Human contain 10 assoicated proteins Bacterial RNase P class A Hairpin ribozyme: Hairpin ribozyme Found in RNA satellites of plant viruses Does not require metal ions for catalyses Species distribution Tobacco Ringspot Virus (TRSV) Chicory Yellow Mottle Virus (CYMV) Arabis mosaic virus (ARMV) Mammalian CPEB3 ribozyme: Mammalian CPEB3 ribozyme Self-cleaving non-coding RNA Located in second intron of CPEB3 gene regulating Messanger RNA polyadenylation Found only in mammales Hammerhead ribozyme: Hammerhead ribozyme Discovered in small RNA satellites of small viruses (1986) Replication by rolling circle mechanism RNA self cleave via a small conserved secondary structural motif termed a hammerhead Cleavage is autocatalytic and takes place in the absence of protein enzymes

Slide 15: Species distribution: Peach latent mosaic viroid Eggplant latent viroid Avocado sunblotch viroid Velvet tobacco mottle virus Satellite RNA Schistosoma mansoni Satellite DNA Dianthus caryophyllus viroid-like DNA Cherry small circular viroid-like RNA Dolichopoda cave cricket Chemistry of Catalysis Breaks substrate strand of RNA at C17, the cleavage site of nucleotide Carries out a isomerisation reaction of Phosphodiester bond Slide 16: Hammerhead ribozyme (type I) Type: Gene;ribozyme; 2 structure: Published; PubMed Seed alignment: Bateman A Avg length: 46.30 nucleotides Avg identity: 70.00% Hammerhead ribozyme (type III) Type: Gene; ribozyme; 2 structure: Published [7] Seed alignment: Bateman A Avg length: 54.2 nucleotides Avg identity: 77% Therapeutic applications : Therapeutic applications Synthetic RNAs containing sequences complementary to the mutant SOD1 mRNA and sequences necessary to form the hammerhead catalytic structure are being studied as a possible therapy for amyotrophic lateral sclerosis . Work is also underway to find out whether they could be used to engineer HIV resistant lines of T-cells. The therapeutic use of trans -cleaving hammerhead ribozymes has been severely hampered by its low-level activity in vivo . The true catalytic potential of trans -cleaving hammerhead ribozymes may be recouped in vivo and therapeutic derivatives are likely to complement other nucleic acid hybridizing therapeutic strategies. Already there are hammerhead ribozymes which are close to clinical application. Group I & GroupII intron ribozyme : Group I & GroupII intron ribozyme Ribosomes: Ribosomes Sub-cellular organelles involved in translation process composed of 65% ribosomal RNA and 35% ribosomal proteins active sites are made of RNA, so ribosomes are now classified as " ribozymes " Deoxyribozyme: Deoxyribozyme DNA enzymes or catalytic DNA , or DNAzymes Discovered in 1994 - Yale Professor Ronald R. Breaker Deoxyribozyme assists in lead ion dependent RNA cleaving operations. Deoxyribozymes developed to catalyse DNA phosphorylation, DNA adenylation , DNA deglycosylation , porphyrin metalation , thymine dimer photoreversion and DNA cleavage. Slide 21: Thank You!

Abzyme
From Wikipedia, the free encyclopedia

An abzyme (from antibody and enzyme), also called catmab (from catalytic monoclonal antibody), is a monoclonal antibody with catalytic activity. Molecules which are modified to gain new catalytic activity are called synzymes. Abzymes are usually artificial constructs, but are also found in normal humans (antivasoactive intestinal peptide autoantibodies) and in patients with autoimmune diseases such as systemic lupus erythematosus, where they can bind to and hydrolyze DNA. Abzymes are potential tools in biotechnology, e.g., to perform specific actions on DNA. Enzymes function by lowering the activation energy of the transition state, thereby catalyzing the formation of an otherwise less-favorable molecular intermediate between reactants and products. If an antibody is developed to stabilize a molecule that's similar to an unstable intermediate of another (potentially unrelated) reaction, the developed antibody will enzymatically bind to and stabilize the intermediate state, thus catalyzing the reaction. A new and unique type of enzyme is produced.

[edit]HIV

treatment

In a June 2008 issue of the journal Autoimmunity Reviews,[1] researchers S Planque, Sudhir Paul, Ph.D, and Yasuhiro Nishiyama, Ph.D of the University Of Texas Medical School at Houston announced that they have engineered an abzyme that degrades the superantigenic region of the gp120 CD4 binding site. This is the one part of the HIV virus outer coating that does not change, because it is the attachment point to T lymphocytes, the key cell in cell-mediated immunity. Once infected by HIV, patients produce antibodies to the more changeable parts of the viral coat. The antibodies are ineffective because of the virus' ability to change their coats rapidly. Because this protein gp120 is necessary for HIV to attach, it does not change across different strains and is a point of vulnerability across the entire range of the HIV variant population. The abzyme does more than bind to the site, it actually destroys the site, rendering HIV inert, and then can attach to other viruses. A single abzyme can destroy thousands of HIV viruses. Human clinical trials will be the next step in producing treatment and perhaps even preventative vaccines and microbicide

[edit]References

Abzymes
As the name suggest abzymesor catalytic antibody are antibody molecule that have conjugated with enzyme to carry out catalysis of some substrate. The abzymes are discovered nearly three decades ago and now with the advancement in the area of protein engineering they show tremendous possibilities. Important article on Abzymes

<="" a="">What are abzymes ? Production of abzymes Uses of abzymess

What are abzymes ? Abzymes are antibodies with variable regions possessing enzymatic activity. Naturally occurring abzymes have been observed in normal individuals (Eg., antivasoactive intestinal peptide autoantibodies) and individuals with autoimmune problems (Eg. DNAse abzymes in systemic lupus erythematosus). Also, a number of

"artificial enzymes" or "designer abzymes" are being developed with any desired enzyme activity and specificity. Production of abzymes For this reason abzymes are not produced naturally. A catalytic antibody is produced in response to molecules that have a structure similar to the proposed expected transition state of the substrate of the reaction to catalyse which the antibody is sought. The details on Abzymes production Uses of abzymes The words said by Dr. Paul on abzymes,"Unlike regular antibodies, abzymes degrade the virus permanently. A single abzyme molecule inactivates thousands of virus particles. Regular antibodies inactivate only one virus particle, and their anti-viral HIV effect is weaker." So abzymes production is one of innovation and there use in HIV treatment may become a future hope for millions of patien

Structural Biochemistry/Protein function/Abzyme

< Structural Biochemistry

[edit]What

is it?

An abzyme is an antibody that expresses catalytic activity [1]. A single molecule of an antibody-enzyme, or abzyme, is capable of catalyzing the destruction of thousands oh target molecules [1]. The efficiency of abzyme technology could permit treatments with smaller doses of medicines at lower costs than are possible today. An abzyme is used to lower the activation energy of a reaction allowing for the transition state to be possible and the product to be formed. An abzyme are typically artificially made and is made by having the immune system make antibodies that bind to a molecule that resembles the transition state of the catalytic process that the researchers want to emulate. Therefore by creating this antibody, now becoming a catalytic antibody allows for this antibody to act as an abzyme reducing the activation energy of the reaction and allowing for the transition state to occur. Abzymes however do occur naturally in the human body.

[edit]Uses

in Medicine

Abzyme are currently being researched for the possible use against HIV infection. The abzymes could target a specific site on the HIV infected cells that do not mutate and then make the virus inert. This is an on going research project by the University of Texas Medical School.By exploiting the highly specific antigen binding properties of antibodies, experimental strategies have been made to produce antibodies to catalyze that chemical reactions. These abzymes are chosen from monoclonal antibodies which are created by immunizing mice with haptens which mimic the transition states of enzyme-catalyzed reactions. The rate of this reaction is promoted by enzyme catalysts that stabilize the transition state of this reaction, thereby decreasing the activation energy and allowing for more rapid conversions of substrate product
[1]

. To successfully create

abzymes that are complementary in structure to this transition state, mice were immunized with an aminophosphonic acid hapten [1]. The study of catalytic antibodies as a whole has vastly increased current understanding of the mechanisms of enzyme catalysis and represents another step forward in the attempts to create artificially engineered biological enzymes
[1]

[edit]Sources

Ribozyme
From Wikipedia, the free encyclopedia

This article is about the chemical. For the rock band, see Ribozyme (band).

Structure of hammerhead ribozyme

A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to perform a chemical reaction. Many ribozymes are catalytic, but some such as self-cleaving ribozymes are consumed by their reactions. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes cleave one of their own phosphodiester bonds(self-cleaving ribozymes), or bonds in other RNAs. Some have been found to catalyze the aminotransferase activity of the ribosome. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme and the hairpin ribozyme. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymeraseribozyme.[1] Mutagenesis and selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds.[2] The "tC19Z" ribozyme can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.[3] Some ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.[4]
Contents
[hide]

1 Discovery 2 Activity 3 Known ribozymes 4 Artificial ribozymes 5 Applications 6 See also 7 References 8 Further reading 9 External links

[edit]Discovery

Schematic showing ribozyme cleavage of RNA.

Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins,[5] were the only known biological 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 structures.[6] The first ribozymes were discovered in the 1980s by Thomas R. Cech, who was studying RNA splicing in the ciliated protozoan Tetrahymena thermophila and Sidney Altman, who was working on the bacterial RNase P complex. 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-tRNAs. In 1989, Thomas R. Cech and Sidney Altman won the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA."[7] The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell.[8] It had been a firmly established belief in biology that catalysis was reserved for proteins. In retrospect, catalytic RNA makes a lot of sense. This is based on 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? Ribo-Nucleic acids as catalysts circumvents this problem. RNA, in essence can be both the chicken and the egg.[9] In the 1970s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for splicing reaction, he found that 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 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, 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 was made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

[edit]Activity
Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze trans-phenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme.[10] RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the 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 became known as the "RNA world hypothesis" of the origin of life. If ribozymes were the first molecular machines used by early life, then today's remaining ribozymessuch as the ribosome machinerycould be considered living fossils of a life based primarily on nucleic acids. A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein conformation in the manner of a chaperone enzyme.[11] Ribozymes have been shown to be involved in the viral concatemer cleavage that precedes the packing of viral genetic material into virions.[12][13]

[edit]Known

ribozymes

Naturally occurring ribozymes include:

Peptidyl transferase 23S rRNA - Found in all living cells RNase P Group I and Group II introns GIR1 branching ribozyme[14] Leadzyme - Although initially created in vitro, natural examples have been found Hairpin ribozyme Hammerhead ribozyme HDV ribozyme Mammalian CPEB3 ribozyme VS ribozyme glmS ribozyme CoTC ribozyme

[edit]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 that have good enzymatic activity have been produced. Tang and Breaker[15] 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. The techniques used to create artificial ribozymes involve Darwinian 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 mutagenic PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are 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 developed an RNA enzyme system capable of self replication in about an hour. By utilizing molecular competition (in vitro evolution) of a candidate enzyme mixture, a pair of RNA enzymes emerged, in which each synthesizes the other from synthetic oligonucleotides, with no protein present. [16]

[edit]Applications
A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection.[17][18]

[edit]See

also

Ribozymes Until about 20 years ago, all known enzymes were proteins. But then it was discovered that some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules). Catalytic RNA molecules are called ribozymes.
Most classes of RNA

Ribonuclease P Group I Introns Group II Introns Spliceosomes Viroids An RNA World? o The Ribosome is a Ribozyme o RNA polymerization by RNA Ribozymes for Human Therapy

transfer RNA (tRNA) ribosomal RNA (rRNA) messenger RNA (mRNA)

are transcribed as precursors that are larger than the final product. These precursors often contain

"head" (5') and "tail" (3') sequences intron sequences

that must be removed to make the final product. Some of the processing steps employ other RNA molecules (always associated with proteins).
Ribonuclease P

Almost all living things synthesize an enzyme called Ribonuclease P (RNase P) that cleaves the head (5') end of the precursors of transfer RNA (tRNA) molecules. In bacteria, ribonuclease P is a heterodimer containing

a molecule of RNA and one of protein

Separated from each other, the RNA retains its ability to catalyze the cleavage step (although less efficiently than the intact dimer), but the protein alone cannot do the job.
Group I Introns Some ribosomal RNA (rRNA) genes

in the mitochondrial genome of certain fungi (e.g., yeast) in some chloroplast genomes in the nuclear genome of some "lower" eukaryotes, for example o the ciliated protozoan Tetrahymena thermophila) o the plasmodial slime mold Physarum polycephalum

contain introns that must be spliced out to make the final product. The splicing reaction is self-contained; that is, the intron with the help of associated proteins splices itself out of the precursor RNA.

Once excision of the intron and splicing of the adjacent exons are completed, the story is over. In other words, although the action is catalyzed by the RNA, only a single molecule of substrate is involved (unlike protein enzymes that repeatedly catalyze a reaction). However, synthetic versions of Group I introns made in the laboratory can in vitro act repeatedly; that is, like true enzymes. The DNA of some Group I introns includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome.
Link to a discussion of transposons.

All the Group I introns share a characteristic secondary structure and mode of action that distinguishes them from the next group.
Group II Introns Some messenger RNA (mRNA) genes

in the mitochondrial genome of yeast and other fungi (encoding the proteins cytochrome b and subunits of cytochrome c oxidase) in some chloroplast genomes

also contain self-splicing introns. Because their secondary structure and the details of the splicing reaction differ from the rRNA introns discussed above, these are called Group II introns. The DNA of some Group II introns also includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome.
Spliceosomes

Spliceosomes remove introns and splice the exons of most nuclear genes. They are composed of 5 kinds of small nuclear RNA (snRNA) molecules and a large number of protein molecules. It is the snRNA not the protein that catalyzes the splicing reactions.

The molecular details of the reactions are similar to those of Group II introns, and this has led to speculation that this splicing machinery evolved from them.
Viroids Viroids are

RNA molecules that infect plant cells as conventional viruses do, but o are far smaller (one has only 246 nucleotides) o are naked; that is, they are not encased in a capsid. Some viroidlike molecules get into the cell as passengers inside a conventional plant virus. These are called virusoids or viroidlike satellite RNAs. In both cases, the molecules consists of o single-stranded RNA whose o ends are covalently bonded to form a circle. o There are several regions where base-pairing occurs across adjacent portions of the molecule. New viroids and virusoids are synthesized by the host cell as long precursors in which the viroid structure is tandemly repeated. These repeats must be cut out and ligated to form the final product. Most virusoids and at least one viroid are self-splicing; that is, they can cut themselves out of the precursor and ligate their ends without the aid of any host enzymes. Thus they represent another class of ribozyme.

Both viroids and virusoids are responsible for a number of serious diseases of economically important plants; e.g. the coconut palm and chrysanthemums. (The problem is so severe with chrysanthemums that all growers in the U.S. now secure their stock from a few companies that raise the plants in "clean" rooms using stringent precautions to prevent infection by the viroid.)
An RNA World? The discovery that RNA molecules can act as catalysts provides a possible solution to a long-standing dilemma:

DNA encodes the genetic information of proteins but DNA replication and transcription requires proteins.

So which came first in the evolution of life? But if RNA can serve both as a

repository of information (in its sequence of nucleotides) and as a catalyst,

then it has both properties needed for life. This provides the basis for the notion that life began as RNA the so-called "RNA World". However, all the reactions described above

involve RNA acting on RNA (not protein) and (except for Ribonuclease P) are self-limited.

Is there evidence that RNA can catalyze the synthesis of proteins? Yes, the ribosome turns out to be a ribozyme. The Ribosome is a Ribozyme Ribosomes are huge aggregates containing 3 (4 in eukaryotes) rRNA molecules and scores of protein molecules. The three-dimensional structure of the large (50S) subunit of a bacterial ribosome was published in August 2000. It clearly shows that formation of the peptide bond that links each amino acid to the growing polypeptide chain is catalyzed by the 23S RNA molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the three-dimensional structure of the RNA.
Link to discussion of ribosome structure and function.

RNA polymerization by RNA In today's world, RNA polymerases made of protein make the RNA molecules (using the antisense strand of DNA as a template [View]). Could RNA alone have done it? It can be done in the laboratory. Wochner, A. et al. report in Science, 332:209, 8 April 2011, their creation of a synthetic RNA molecule that when presented with singlestranded RNA templates, polymerizes ribonucleotide triphosphates into strands of RNA complementary to the template. Their synthetic RNA polymerase was able to faithfully incorporate up to 95 nucleotides into complementary strands of RNA. One product was a functional endonuclease ribozyme.

Ribozymes for Human Therapy

The ability of ribozymes to recognize and cut specific RNA molecules makes them exciting candidates for human therapy. Already, a synthetic ribozyme that destroys the mRNA encoding a receptor of Vascular Endothelial Growth Factor (VEGF) is being readied for clinical trials. VEGF is a major stimulant of angiogenesis, and blocking its action may help starve cancers of their blood supply.
Link to discussion of angiogenesis inhibitors and cancer.

Ribozymes are antisense RNA molecules that have catalytic activity. They function by binding to the target RNA moiety through Watson-Crick base pairing and inactivate it by cleaving the phosphodiester backbone at a specific cutting site. Five classes of ribozymes have been described based on their unique characters in the sequences as well as three-dimensional structures (Bunnell,1997). They are denoted as (1) the Tetrahymena group I intron, (2) RNase P, (3) the hammerhead ribozyme, (4) the hairpin ribozyme, and (5) the hepatitis delta virus ribozyme. They may catalyze self-cleavage (intramolecular or "in-cis" catalysis) as well as the cleavage of external substrates (intermolecular or "in-trans" catalysis) (Ohkawa, 1995). The leading study was conducted by Thomas Cech on ciliated protozoan Tetrahymena Thermophila group I introns in 1980s, which showed that RNA can participate in the intramolecular catalysis of the self-splicing and acts as a protein enzyme. The definition of biological enzyme was broadened since then, by recognizing the enzymatic function of some RNA molecules.

Figure 1. Tetrahymena Group I intron secondary and tertiary structures (Golden B., Cech T.)

P4-P6 Structure (Doudna J. , Cech T.)

Figure 2. A hammerhead ribozyme structure with magnesium cation binding sites (Murray,1996) The name of hammerhead ribozyme is given by the similarity between its secondary structure and the shape of a hammerhead. They are the best understood subcategory of all ribozymes. As well as other ribozymes, the hammerhead ribozyme is an antisense RNA. Some of the ribonucleotides within the sequence selectively form Watson-Crick base pairs with others to form a stem, while the rest stay in single stranded state called loop. These loops and stems can be predicted at the secondary structure level using conformational energy analysis, such as RNAdraw and mfold; and three dimensional structures were obtained mainly by X-ray crystallography.

Figure 3. Secondary structures of in cis and in trans hammerhead ribozymes

On the left of Figure 3, the structure of the wild-type, cis-acting hammerhead ribozyme is shown. The three helices and the cleavage site are indicated. The self-splicing happens after the sequence G-U-C between helix I and III, and the G and U are conserved and crucial for the catalysis. Except for itself, a hammerhead ribozyme is not catalytic in cells since its nature of intramolecular reaction (Symons, 1989). However, most ribozymes can be chemically engineered to cleave RNA in trans by separating the catalytic domain from ribozyme and attaching recognition (i.e., antisense) arms to the catalytic center, in order to target to a substrate. These trans-acting ribozymes can be very useful in the study of molecular biology and pharmaceutics. In comparison to the conventional antisense RNAs, ribozymes provide the potential of turnover, with a single molecule being able to inactivate multiple target RNAs. Another important property of ribozyme is the specificity,

Figure 4. Turnover cycle of RNA cleavage by a hammerhead ribozyme. Binding of the enzyme and substrate results in the catalytically active structure. After cleavage, the two product strands dissociate and the ribozyme strand can go into the next cycle. which means the fidelity to cleave a unique target. Both of turnover and specificity are affected by binding arm length (helix I and III). If the length of binding arms is very short, the rate of dissociation of the target from the ribozyme may exceed the rate of cleavage, resulting in poor efficiency

(Rossi, 1997). However, stable hybrids exhibit low catalytic activity because of slow dissociation of the cleaved substrate (Bertrand, 1994). Thus, the ideal situation is to have arm lengths that aid cleavage, yet provide for quick dissociation of the cleaved products. Read more about hammerhead ribozymes in structural biology, catalysis, application and valuable r