Introduction
One approach to engineer an enzyme with novel catalytic property is to manipulate a molecule
directly. Understanding the relationship between various structures and their functions
would enable us to design a molecule like a machine. Another approach is to rely upon
evolution to generate a molecule with a specific property. In vitro selection
or SELEX (Systematic Evolution of Ligands by EXponential
enrichment) has been used to engineer RNA molecules with various functions. In theory,
in vitro selection does not require detailed knowledge of the target molecule.
In in vitro selection, three important processes
must be carried out:
1) Introduction of genetic variations or mutations
2) Selection of variant molecules best suited for a particular process
3) Amplification of the selected molecules.
Thus, a detailed knowledge of structure -- either of the RNA or of the
target -- is not needed for this process.
There are several applications for in vitro evolution:
1) engineering an enzyme with a novel function; 2) investigating the RNA world hypothesis;
3) designing molecules for clinical applications. In theory, RNA molecules that bind
specific ligands can be designed for clinical or diagnostic purposes. Compared to production
of monoclonal antibodies, in vitro process could be carried out more easily. Ribozymes
can be modified to cleave infectious viral or bacterial components. According to the
RNA world hypothesis, the primitive self-replicating system was composed of ribonucleic
acids and the processes that are carried out by protein enzymes today must have
been carried out by RNAs. In vitro evolution has been used to investigate the theory
and its ultimate goal is to create a RNA replicase that is composed entirely of RNA.
General procedure
The general procedure for in vitro evolution or SELEX is shown in the figure.
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Figure 11. The basic cycle of in vitro evolution of RNA.
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The process starts with a population of DNA templates. The starting DNA templates
can be oligonucleotides with random sequences or oligonucleotides complementary to a
known ribozyme with various mutations. The DNA templates are transcribed into RNAs by
T7 RNA polymerase. From a pool of RNA molecules, a fraction of the RNA molecules are
selected based on their ability to carry out specific function. The selected RNA molecules
are copied into cDNAs by reverse transcriptase. PCR (Polymerase Chain Reaction) is used
to amplify the cDNAs. Genetic variations or mutations can be introduced to a population
of DNA templates at a desired cycle. If the process is repeated for a number of cycles,
the resulting population of RNAs must be able to carry out a specific function more efficiently
than the starting pool of RNAs.
Specific examples
The most critical step in in vitro evolution is the selection
step. The selection step must be designed so that it is possible to distinguish RNAs
that carry out a specific function from those that do not.
A. Use of an existing ribozyme as template
In vitro evolution can be used to modify the catalytic mechanism
of an existing ribozyme. In normal biological system, Tetrahymena ribozyme is
a self-splicing group I intron that catalyzes excision of its own sequence from a precursor
rRNA by the sequence-specific phosphoester transfer reaction. The ribozyme has a limited
ability to cleave DNA at a high temperature (50° C). Beaudry and Joyce (1992) reported
the experiment in which the in vitro evolution was used to engineer ribozymes that specifically
cleave DNA at a physiological temperature (37° C). The starting pool was consisted
of the Tetrahymena ribozymes with random mutations introduced at the catalytic
core. The selection scheme was based on the ribozyme's ability to cleave a DNA substrate
and attach a portion of the DNA substrate to the ribozyme's 3'-end. The selection was
achieved during cDNA synthesis in which a primer was designed to bind the junction where
the DNA substrate had been attached. The primer did not bind to unreacted molecules.
At each amplification stage, PCR was used under mutagenic condition to generate a population
of heterogeneous molecules. The cycle was repeated 10 times.
B. Aptamers - RNA molecules that bind specific ligands
RNA molecules that bind specific ligands are called "aptamers"
(from the Latin word 'aptus', to fit). Selection of aptamers that bind small dyes was
reported by Ellington and Szostak (1990). The starting pool were consisted of DNA molecules
made up of 100 nucleotides of random sequences. The question they asked was how many
of these random RNA molecules would fold in a specific manner to bind specific ligands
- small dyes in this case. The selection scheme was based on the RNA molecules' ability
to bind affinity columns with covalently attached dyes. A fraction of the RNA molecules
that did not bind the dyes were simply washed out from the columns. The selected aptamers
can recognize specific ligands and have no recognizable sequence similarities between
them.
C. Selection of new ribozymes from random sequences
Bartel and Szostak (1993) successfully selected new ribozymes
from a pool of 300 nucleotide-long(nt) RNAs of mostly random sequences (this amounts
to approximately 1015 different molecules). The selected ribozymes catalyze
the reaction in which the ribozymes ligate substrate oligoribonucleotides to their own
5'-end. This reaction is similar to the chain elongation during RNA polymerization in
which the 3'-OH of an elongating strand attacks the a-phosphate
of an adjacent 5'-triphosphate, displacing a pyrophosphate (PPi) and yielding 3',5'-phosphodiester
bond. The initial RNA pool were consisted of random 220 nt RNAs, that were flanked
by 40 nt 5'- and 3'- constant regions. The 5'-constant region was designed to anneal
with the substrate oligonucleotide, placing the substrate in a position where the reaction
can take place. Error-prone PCR was used to introduce mutations in the central core of
random sequences. The selected ribozymes exhibited the reaction rate that is 7 million
times faster than the uncatalyzed reaction.
The rate enhancement achieved by the new ribozymes is not comparable to
the one by RNA polymerase. However, it should be noted that it is not possible to screen
all possible sequence combinations. Technically, the experiment must be carried out in
a condition that no aggregation of RNA molecules takes place. Although it is not possible
to examine all possible sequence combinations, these experiment suggests that simple
systems can be created by screening relatively large sequence space.
In vitro evolution
1. Bartel D. P. and Szostak J. W. Isolation of new ribozymes from a large pool of random
sequences. Science (1993) 261, 1411-1418.
2. Beaudry, A. A. and Joyce, G. F. Directed evolution of an RNA enzyme.
Science (1992) 257, 635-641.
3. Benner S. A. Catalysis: Design versus selection. Science (1993)
261:1402-1403.
4. Ellington, A. D. and Szostak, J. W. In vitro selection of RNA molecules
that bind specific ligands. Nature (1990) 346:818-822.
5. Illangasekare, M., Sanchez, G., Nickles, T., and Yarus, M. Aminoacyl-RNA
synthesis catalyzed by an RNA. Science (1995) 267:643-647.
6. Noller, H. F., Hoffarth, V., and Zimniak, L. Unusual resistance of peptidyl
transferase to protein extraction procedures. Science (1992) 256:1416-1419.
7. Piccirilli, J. A., McConnell, T. S., Zaug, A. J., Noller, H. F., and
Cech, T. R. Aminoacyl esterase activity of the Tetrahymena ribozyme. Science
(1992) 256:1420-1424.
8. Wright M. C. and Joyce, G. F. Continuous in vitro evolution
of catalytic function. Science (1997) 276:614-617.
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Last updated: 3:11pm on 9/26/08 by pgegen@ku.edu