There are many different types
of ribozymes in nature but our lab has focused upon the hammerhead motif which is found as part of the self - cleaving domain of small plant viruses.
Two years ago, for example, researchers in the United Kingdom reported isolating an
RNAP ribozyme capable of stitching together RNAs up to 200 nucleotides long, again when matching them up to a template strand.
Welch et al. «Expression of
Ribozymes in Gene Transfer Systems to Modulate Target RNA Levels», Current Opinion in Biotechnology, 9: 486 - 496, 1998.
The contribution made by individual functional groups of the G.U pair in the context of the
Tetrahymena ribozyme was examined by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex.
To date 10 HIV positive patients have been treated
with ribozymes developed in the lab.
After only 11 «generations» of evolution, Unrau and Bartel had
produced ribozymes highly effective at catalyzing nucleotide formation.
There are already some companies dedicated to bringing
ribozymes from the laboratory to the commercial world, with potential applications as sensitive sensors of biowarfare germs or as medical diagnostic tests.
RNA organisms could evolve
new ribozymes as well and also produce them in bulk as they multiplied.
C. Schuabb, N. Kumar, S. Pataraia, D. Marx, R. Winter Pressure modulates the self - cleavage step of the
hairpin ribozyme Nature Communications 8 (2017), 14661 DOI: 10.1038 / ncomms14661
The discovery in the early 1980s that some RNA molecules,
called ribozymes, can catalyze chemical reactions led many biologists to speculate that RNA was the primordial biomolecule.
The
sunY ribozyme and its small selected variant can both catalyze template - directed oligonucleotide assembly.
A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the
original ribozyme under the selection conditions.
«We studied in detail, by reproducing the reaction conditions, the splicing process carried out by the group II
intron ribozyme,» explains Lorenzo Casalino, SISSA PhD student and first author of the study.
Several genetic approaches have been taken to reduce or eliminate CCR5 expression in human cells, including the use of ribozymes [17], [18], single - chain intracellular antibodies [19], trans - dominant coreceptor mutants [20], and RNAi [21], [22].
Ribozymes developed in our research program have been developed to target HIV and these have been inserted into human blood progenitor cells via a retroviral vector as part of a gene therapy program for the treatment of HIV infection.
In particular, at some point some of the RNA sequences mutated,
becoming ribozymes that sped up the copying of RNA — thus adding a competitive advantage.
Ribozymes derived from the self - splicing pre-ribosomal RNA of Tetrahymena act as sequence - specific endonucleases.
These results indicate that P4 and P6 are coaxial in the P4 - P6 domain and, therefore, in the
native ribozyme.
To see
if ribozymes could evolve this ability, biochemists Peter Unrau and David Bartel at the Massachusetts Institute of Technology in Cambridge began with a pool of more than 1015 different RNA molecules.
The discovery of
ribozymes not only changed our understanding of how life works today, but it also offered insights into the origin of life itself.
In subsequent work at the Whitehead Institute and MIT, Bartel modified this type of
ribozyme through tinkering and evolution.
Today both Bartel and Szostak keep students and postdocs busy in their labs evolving
improved ribozymes that can build longer copies.
Watching ribozymes at work revealed how primordial RNA could store genetic information and act like an enyzme.
Other ribozymes have shown promise in fighting cancer, heart disease, and HIV.
Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena
thermophila ribozyme molecules.
They tested it in a rat model of ADRP and found that after 2 to 3 months, eyes that had been injected with the virus -
ribozyme combo contained 30 to 40 % more rod cells than eyes that received a dummy injection.
The therapy uses a
designer ribozyme, a short strand of RNA that chops up other RNA, to seek and destroy mutant RNA before it can be used to build a protein that kills the eye's rod cells.
This
engineered ribozyme is able to function effectively both as a catalyst and as a template in self - copying reactions.
This biologically active RNA fold is the same as that found previously for two modified
hammerhead ribozymes.
As a test of this idea, two retroviral vectors — one encoding a hammer -
head ribozyme designed to cleave lacZ transcripts and another encoding the lacZ messenger RNA — were coexpressed inside retroviral packaging cells.
Cellular compartmentalization of RNAs is thought to influence their susceptibility to
ribozyme cleavage.
If the RNAP
ribozyme successfully created a new RNA, the new strand would signal that by binding to a specific molecular target in its vial.
The structure indicates the extent of RNA packing required for the function of
large ribozymes, the spliceosome, and the ribosome.
This toolkit includes TALEN and CRISPR nucleases, nickases, and gene activation systems as well as Cys4, tRNA, and
ribozyme systems for multiplex gRNA expression.
They started by synthesizing a large library of DNA strands intended to encode the starting
RNAP ribozyme.
By this directed evolution we were able to
produce ribozymes that can catalyze the copying of relatively short strands of other RNAs, although they fall far short of being able to copy polymers with their own sequences into progeny RNAs.
In a world before DNA, RNA molecules would have had to be a lot more accomplished than the
Tetrahymena ribozyme.
This notion has inspired several experiments, both at our lab and at David Bartel's lab at the Massachusetts Institute of Technology, in which we «evolved»
new ribozymes (an RNA molecule that can act as an enzyme).
We have studied the correlation between structural dynamics and function of the
hairpin ribozyme.
In several simpler organisms, this key process is carried out by group II introns, enzymes entirely made up of RNA (different from the true protein enzymes)
called ribozymes that are able to self - cleave by removing themselves from the mRNA filament and thereby promoting RNA maturation.
The
sunY ribozyme is derived from a self - splicing RNA group I intron.
The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the
original ribozyme, of its rate of self - copying.
A handful of
ribozymes in a beaker — no matter how accomplished they may be — simply doesn't make the cut.
Efforts to improve catalytic efficiency and co-localization
of ribozymes with their targets in mammalian cells are still an ongoing effort in the lab.
You may wish to start by looking at the work of miller and urey, followed by some reading on prions and
ribozymes.