This document provides an overview of ribozymes, including their definition, classes, history of discovery, mechanisms of action, design, delivery methods, and therapeutic applications. Key points include: Ribozymes are RNA molecules with enzymatic catalytic activity; There are two main classes - self-cleaving and self-splicing ribozymes, which include hammerhead, hairpin, hepatitis delta virus, group I and II introns, and RNase P; They were discovered in the 1980s and shown to catalyze critical reactions without protein enzymes; Therapeutic applications aim to target disease-related genes and are showing promise for cancer, infectious diseases, and genetic disorders in clinical trials.

1. By
Fatma Hussien Fahmy
Under supervision of
Dr.Maher A.Kamel
Dr.Rasha Eltahhan
Biochemistry dep. (MRI(

2. • Defenition of ribozyme
• Catalytic Activities of Ribozymes
• history of Ribozymes
• Classes of Ribozymes
• Production of Ribozymes
• Modification of ribozymes
• Delivery of Ribozymes to Cells
• Therapeutic applications of ribozymes

3. • A ribozyme (ribonucleic acid enzyme) is
an RNA molecule that is capable of
performing specific biochemical reactions,
similar to the action of protein enzymes.

4. • Ribozymes are RNA molecules endowed with catalytic activity
and capable of cleaving mRNA molecules in a sequence specific,
catalytic manner. They contain sequences for selective ligation
with target mRNAs which confers upon them high specificity.
They also contain sequences that perform cleavage reactions
with the target mRNA. By modifying the substrate recognizing
sequences, ribozymes can be specifically tailored for the
suppression of particular genes.

6. • All known enzymes were considered
as proteins until the discovery of
some RNA molecules that can act as
enzymes in 1980s.
• Thomas Cech, at the University of
Colorodo , 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.

7. • Shortly thereafter,
Altman's group, at Yale
University, showed that
the RNA component of
RNase P, M1 RNA, from
Escherichia coli was
likewise able to process
tRNA precursors
without any protein co-
factors. Czech and
Altman shared the
Nobel Prize in
Chemistry in 1989 for
this work.

8. natural ribozymes can be classified into two
different groups:
1) the self-cleaving RNAs which include the
hammerhead, hairpin, hepatitis delta virus, Varkud
satellite .
2) the self-splicing ribozymes that are the group
I and II introns, RNase P.

9. The first hint of catalytic activity in RNA molecules concerned a group I intron
found in the precursor of Tetrahymena thermophila large subunit rRNA.
Group I introns have also been found to interrupt a large variety of tRNAs,
mRNAs and rRNAs in bacteria and many other organisms.
The Tetrahymena thermophila
precursor rRNA contains a group
I intron capable of catalyzing its
own excision. Self-splicing of the
intron requires presence of a
guanosine cofactor and a divalent
cation, either Mg2+ or Mn2+, and
occurs via two sequential
transesterification reactions.

10. •Group I intron ribozyme may prove to be an important tool for RNA-
directed therapy.
•It can be specifically designed to repair abnormal mRNA molecules.
• It has been demonstrated that modified group I introns are
suitable for the correction of deficient mRNAs.
• For example, a trans-active group I intron was used to repair
mutant β-globin RNA in erythrocyte precursors from patients with
sickle cell anemia by replacing the mutated part of the β-globin RNA
by the γ-globin-3’- exon.
• Additional attempts have been reported to address diseases caused
by trinucleotide repeat expansions, including Huntington’s disease
and myotonic dystrophy.

11. • Group II introns have been found in bacteria and in the
mitochondrial and chloroplast genomes of fungi, plants, and yeast
• They don’t require guanosine residue.
• Multidomain metalloenzyme
• Less widely distributed than
group I.
Mechanism:
The 2’OH of a specific adenosine acts as
a nucleophile and attacks the 5’ splice
site creating a branched intron
structure. The 3’ OH of the 5’ exon
attacks the 3’ splice site, ligating the
exons and releasing the intron as a lariat
structure.

12. • Ribonuclease P (RNaseP), a ribonucleoprotein, is an essential tRNA
processing enzyme found in all living organisms. Since its discovery almost
40 years ago, research on RNase P has led to the discovery of the catalytic
properties of RNA.
Mechanism:
• All RNase P enzymes are ribonucleoproteins [bacteria: 1RNA + 1 protein subunit;
eukaryotes: 1 RNA + many protein subunits (11 in human)],
• In Ribonulease – P, protein component facilitates binding between RNase and
t-RNA substrate.
• Requires divalent metal ions (like Mg2+) for its activity.
• Endo-ribonuclease responsible for generating 5’ end of matured tRNA molecules.
• Cleavage via nucleophilic attack on the phosphodiester bond leaving a 5’-phosphate
and 3’-hydroxyl at the cleavage site.

13. • Hammerhead ribozymes (HHRZs) are tiny 30 nucleootides
autocatalytic RNAs that cleave single-stranded RNA. They are found in
nature as a part of certain virus-like elements called virusoids
• The HHRZ is so named because its secondary structure is similar to
that of a hammer head, but actually its tertiary structure is more like
‘Y’ shaped.
They have been used to cleave many RNA
targets in vitro; however, there has been
limited success in ribozyme-mediated gene
inactivation in vivo. A major problem that
researchers face when aiming to apply
hammerhead ribozymes intracellularly for
therapeutic purposes is the requirement of
elevated concentrations of divalent ions to
obtain high catalytic activity.

14. • The hairpin ribozyme is an RNA motif
that catalyzes RNA processing
reactions essential for replication of
the satellite RNA molecules in which
it is embedded.
• It does not require direct
coordination of metal cations to
phosphate.
• These ribozymes have been
developed mainly for antiviral
applications

15. •The hepatitis delta virus (HDV) ribozyme is found in a satellite
virus of hepatitis B virus, a major human pathogen.
•The hepatitis delta virus (HDV) ribozyme is a non-coding RNA
found in the hepatitis delta virus that is necessary for viral
replication and is thought to be the only catalytic RNA known to
be required for viability of a human pathogen.
•The ribozyme acts to process the RNA transcripts to unit
lengths in a self-cleavage reaction.

17. Ribosome is a large and complex molecular
machine, found within all living cells, that
serves as the primary site of
(translation).
It consists of two sub-units, one large and
one small. The large(50s) subunit has 5s
and 23s rRNAs as its core.
After the determination of the high-
resolution structure of ribosome, it was
clear that the 23s subunit is responsible
for the catalytic peptidyl
transferase activity that links amino acids
together.
That is why ribosome is also a ribozyme.
The red region indicates the site where
mRNA interacts with the tRNA
anticodons.

18. •For designing a ribozyme, one first selects a region in the
target RNA containing NUX, where N stands for any nucleotide,
U represents uridine and X is any nucleotide except guanosine.
• Two arms of antisense nucleotides 6-8 nucleotides long that
flank the 21 nucleotide sequence forming the catalytic
hammerhead between them are then designed based on the
target sequence surrounding the third nucleotide (X) of the
target triplet.
•Hairpin ribozyme design employs the same approach except
that the catalytic core is larger (34 nucleotides) and the
ribozyme targeting domains require more specificity. Hairpins
recognize the sequence NNYNGUCNNNNNN, where N is any
nucleotide and Y is a pyrimidine. The underlined target bases
(NGUC) are not base-paired with the ribozyme.

20. Synthetic ribozymes comprised of all ribonucleotides are
susceptible to endo- and exonuclease breakdown. To overcome the
stability, substrate specificity and cell permeability limitations
imposed by all-ribonucleotide ribozymes, much effort has been
devoted to the chemical modification of ribozymes while
maintaining their ability to cleave substrate mRNA.
Some of the most common modified ribozymes are discussed as
follows;
1.Catalytic Antisense RNAs
2.Retroviral Nucleocapsid Proteins
3.Allosteric Ribozymes

21. Delivery of genes encoding
ribozymes (i.e. endogenous delivery of
ribozymes) using viral gene therapy
vectors is the principal alternative to
direct delivery of ribozymes (i.e.
exogenous delivery of ribozymes) to
tissues for inhibition of gene
expression.
The most widely used viral vectors
are the retroviral vectors, adenovirus
vectors and adeno-associated virus
vectors. The viral vectors have been
modified to eliminate their toxicity
and maintain their high gene transfer
capability.
mainly used for exogenous delivery
of ribozymes.
Although their low efficiency
remains a major drawback, non-viral
vectors have many advantages over
viral vectors, including ease of
production, lower toxicity and
immunogenicity and lower cost
.Moreover, non-viral vectors
provide various means of physical
protection of the ribozymes for
safe delivery into the cells.(from
renal filtration , enzymatic
degradation ,immunoligical )
cationic in nature (lipids , polymers,
liposome )

22. Ribozymes contain sequences for selective ligation with target
mRNAs which confers upon them high specificity. They also contain
sequences that perform cleavage reactions with the target mRNA. By
modifying the substrate recognizing sequences, ribozymes can be
specifically tailored for the suppression of particular genes. A large
variety of gene products that are responsible for different
pathological conditions have been targeted successfully using this
strategy.
ribozymes offer promising potential in the treatment of cancer, infectious
diseases and genetic disorders .Currently, there are few examples of
ribozymes that are under clinical trails for application to human diseases
such as for AIDS and cancer. The first clinical trial using a ribozyme
targeted human immunodeficiency virus 1(HIV-1) had shown that an anti-
HIV-l gag ribozyme that was delivered intracellularly can interfere with
both pre-integration and post-integration events of the HIV replication
cycle, by cleaving incoming viral RNA and transcribed mRNAs.

23. •Only two clinical trials are in progress to evaluate the potential of
chemically modified hammerhead ribozymes to fight cancer.
•The first, ANGIOZYME (Sirna Therapeutics, Inc.), a hammerhead
ribozyme that targets mRNA that encodes VEGF, is being examined in
a phase II trial for treatment of metastatic colorectal cancer.
•The other ribozyme in cancer clinical trials, HERZYME, is of a
class of modified ribozymes (so called Zinzymes) that has high
catalytic activity under physiological Mg2+ conditions and targets the
mRNA that encodes human epidermal growth factor-2, and is in phase
I clinical trials to determine toxicity and efficacy in breast and
ovarian cancer patients.