Ribozyme

Deepti
M.Sc MBT
2nd Semester
 INTRODUCTION
All known enzymes were proteins.
It was discovered that some RNA molecules also have
enzymatic property i.e., catalysing covalent changes in the
structure of substrates.
These ribonucleic acids with enzyme like activity were
named RIBOZYME.
The first ribozyme were discovered in the 1980s by
Thomas R. Cech and Sidney Altman.
These ribozymes were found in the intron of an RNA
transcript, which removed itself from the transcript.
In 1989, Thomas R. Cech and Sidney
Altman won the nobel prize in chemistry for
their “discovery of catalytic properties of
RNA.”
The term “ribozyme” was first introduced
by Kelly Kruger et al. in 1982 in a paper
published in cell.
The few known naturally occurring
ribozymes are :
RNase P
Group I and group II introns
Leadzyme
Hairpin ribozyme
Hammerhead ribozyme
HDV (hepatitis Delta Virus) ribozyme
 Classes of Ribozymes
RNAs are broadly grouped into two classes based on their
size and reaction mechanisms:
Large ribozymes (hundreds of nucleotides) and
small ribozymes (less than one hundred nucleotides).

Large ribozymes Small ribozymes

Group I and II introns Hammerhead ribozyme
Ribonuclease P ribozyme Hairpin ribozyme
HDV ribozyme
 Hammerhead ribozymes
Hammerhead ribozyme is the smallest of all the
naturally occurring ribozymes.
It has been found in several satellite RNAs of plant
viruses and viroids.

The reaction is composed of 3 steps:

1. Binding of the ribozyme to the target
2. Cleavage of the target
3. Release of the cleavage products
Hammerhead ribozyme has an absolute
requirement for divalent metal ions.
Mg2+ is the referred metal, but Mn2+ , CO2+, and
Ba2+ also support cleavage activity.
Standard reaction conditions include pH 7.5 ,at
temperature between 27 degree Celsius and 0
degree Celsius.
The crystal structure of the hammerhead ribozyme
was the first example of an atomic structure of a
catalytic RNA molecule to be determined.
Interestingly, the three helices of the ribozyme are
arranged in a Y shape.
Secondary and tertiary structure of a hammerhead
ribozyme.
Hairpin ribozymes
It is the RNAs domain found in satellite RNAs of
pathogenic plant viruses.
Three hairpin ribozymes have been found in nature,and
the satellite RNA associated with tobacco ring spot
virus (TRSV) is the best characterised.
Other two hairpin motifs isolated from different
satellite viruses show sequence variations.
Like the hammerhead it effectively uses Mg2+ and
Ca2+ ions however it is inhibited by Mn2+ and O2+.
Catalytic activity of hairpin ribozyme results from
distortion of the substrate RNA and general acid-base
catalysis by neighbouring nucleotides without
involvement of metal ions in catalysis.
The condition required for the reaction is pH 7.5 and
temperature between 27-50 degree celcius.
Hairpin ribozyme effectively cleaves a
phosphorothioate at the target site in the presence of
EDTA and inert cobalt (III) complexes.
The hairpin contain four helical regions and two single
stranded loops. It consist of two domains each
harbouring two helical regions separated by an internal
loop and connected by a hinge region.
Hairpin ribozymes have been developed mainly for
antiviral applications.
The two independently folding domains A and
B each consist of two helices (H1, H2 and H3, H4; black lines represent
Watson–Crick base pairing) that flank internal loops A and B,respectively.
Ribozyme nucleotides are numbered 1 to 50.
 Introns
Introns are non coding sequences that interrupt the
coding sequences of most eukaryotic genes.

Group I Introns
Group I introns are the only class of introns whose
splicing acquires a free guanine nucleoside.
Group I introns are found in plant and fungal
mitochondria, bacteriophage, eubacteria and
chloroplast tRNA.
Group I introns have phylogenetically conserved
secondary structures.
Group I introns generally consist of hundreds of
nucleotides.
The enzymatic activity of group I introns involve a
two step transesterification with a requirement for a
divalent cation, such as magnesium and a guanosine
co-factor.
Group II Introns
Group II introns are commonly found in
mitochondrials genes in plants,fungi,yeast and other
lower eukaryotes.
In vitro cleavage requirs high Mg2+ concentration and
monovalent cation such as K+ and under physiological
salt conditions a hydrolytic mechanism, in which a
water molecule is positioned for attack tends to
dominate in vitro analyses.
It has been demonstrated that group II introns can be
redirected to insert themselves into therapeutically
relevant DNA target sites into human cells.
 RNase P
RNase P is a ubiquitous enzyme that acts as an
endonuclease to generate the mature 5’-end of the tRNA
precursors.
In bacteria, RNase P exists as a ribonucleoprotein
complex, consisting of a long RNA, typically 300-400
nucleotides in length and a small protein of
approximately 14kDa.
RNase P can be considered to be the only known true,
naturally occurring trans- cleaving RNA enzyme.
Further RNase P is one of two known multiple turnover
ribozymes in nature, the discovery of which earned
Professor Sidney Altman the Nobel Prize in chemistry in
1989.
It has been shown that human nuclear RNase P is
required for the normal and efficient transcription of
various small non-coding RNA genes, such as
tRNA,which are transcribed by RNA polymerase III.
RNase P is unique in that it consists of both RNA and
protein subunits,but all of the catalytic activity resides
in the RNA subunit.
The protein subunit is necessary for in vivo activity,
but under high Mg2+ and monovalent salt
concentrations the RNA portion itself is sufficient for
catalysis.
 Artificial ribozymes
Artificially produced self-cleaving RNAs that have
good enzymatic activity have been produced.
 Zang and Breaker isolated self-cleaving RNAs by
in-vitro selection of RNAs originating from
random-sequence RNAs.
The technique used to discover artificial ribozymes
involve Darwinian evolution. This approach takes
advantage of RNAs dual nature as a catalyst and as
an informational polymer.
Leadzyme
Leadzyme is a small ribozyme that was artificially
made using in vitro selection techniques.
Leadzyme is able to cleave RNA in the presence of
lead.
The structure of leadzyme has been determined by
X-ray crystallography.
Ligase ribozymes
RNA ligase ribozyme were the first of several
types of synthetic ribozymes produced by in vitro
evolution and selection techniques.
They are an important class of ribozyme because
they catalyse the assmebly of RNA fragments into
RNA polymers by forming phosphodiester bond.
 Applications
 Ribozymes as therapeutic agents
They have been proposed for treatment of a variety
of diseases including infectious diseases and cancer.
The first step to use ribozyme as therapeutic agent
is designing the ribozyme in which critical
properties such as specificity and turnover should
be considered.
After designing the second step is delivery of
catalytic oligonucleotides itno the desired
subcellular compartment of the targeted cell
population within an population.
Advantages of ribozyme in clinical
application
Ribozymes including hammerhead ribozymes, can be
used in the study of gene function and gene therapy for
diseases.
The property of ribozyme makes them suitable in the
research of genetics and developmental biology.
The other important application of ribozyme is to
develop new drugs and protocols for gene therapy.
By protecting cells against viral expression cytopatheic
effects, ribozymes may promote survival of
populations of functional transduced cells in
vivo,leading to increased therapeutic effects over time.
By producing only therapeutic RNAs, ribozymes are
likely to avoid entirely or at least largely the
problems of immune eradication of transduced cells
or production of autoimmune antibodies.

Clinical trials with ribozyme

Several clinical studies with ribozymes have been
carried out to treat infectious diseases or cancer.
Tumours are rapidly growing tissues with a high
demand for oxygen and nutrition.therefore the
formation of new blood vessels, a process called
angiogenesis, is required for sustained tumour
growth.
The furthest developed , chemically synthesised
ribozyme “ANGIOZYME” was designed to inhibit
tumour angiogenesis.
HERZYME is the ribozyme that was studied in a
clinical trial by RPI. It is directed against the mRNA
of the human epidermal growth factor receptor-2
(HER-2), which is over expressed in aggressive
breast cancer. This ribozyme was based on the
chemistry developed by Zinnen et al.
Ribozymes- molecular scissors for
investigating gene function
Ribozymes are molecular scissors that cut RNA, the
molecular messages given by genes in order to
produce proteins.
These molecular scissors provide a very useful means
of studying gene function since by cutting the RNA
with a ribozyme a gene can be effectively turned off.
Thank you