|
The directed molecular evolution aims to harness the principle of survival of the fittest - at a microscopic level in a test tube rather than in the jungle, and in week rather than in millenniums and thus currently provides the best means to study how Mother Nature engineers and ‘evolve’ the Life. When exploited in a laboratory under controlled and repeating cycles of mutation, selection and amplification, it enables to ‘evolve’ novel bio-molecules in desired directions. In recent, directed evolution has been greatly aided by introducing genotype-phenotype linking technologies such as RNA-protein fusion (originally described in our laboratory by Prof Husimi and coworkers), in which coding mRNA and/or DNA part (genotype) of molecule is covalently linked to its encoded protein (phenotype), using puromycin as an adaptor molecule and thus united in a single molecule. Next, affinity selection via the protein moiety can determine the genetic information of selected functional molecule via the mRNA moiety and thus enables facile synthesis and selection of peptide and protein libraries of more than trillions of different sequences in test tube. Below is a brief description of some (but not least) areas of ongoing research centered on directed molecular evolution:
An artificial evolution of
ribosome-mRNA interaction by searching new translational regulatory
sequences (5’UTR)
Following the completion of insect, plant, and human genome decoding (sequence information) projects, we are now in the ‘post-genomic’ era, where attention has turned to analyze the structure and function of protein abundance, under the name of proteomics (functional information). However, high-throughput profiling of the entire proteome is based on efficient expression of the genome encoded proteins and thus on the availability of sufficient amount of synthesized active proteins. Cell-based translation systems have been unable to meet the current challenges where the translation initiation is finely regulated by 5’-capped and 3’-poly(A) containing long untranslated regions (UTRs). Cell-free systems, in contrast, are becoming the favored alternative to cell-based methods. However, the efficiency of these systems is reputed to be unstable and restricted due to the problematic of maintaining long UTRs at the both ends of translated region. In addition, long leader sequences impose a burden on cell-free gene expression: first by hampering the cap-dependent ribosomal scanning on long 5’UTR, containing AUGs and secondary structure and second, by providing substrate for the mutational origin of premature translation-initiation sites and thus enhancing the rate of acquisition of a premature translation-initiation site, which generally result in defective proteins. Therefore, in this research, in order to potentially improve the cell-free system (in particular to eliminate the 5’-cap, which is substrate of decapping enzymes and 3’-poly(A) dependency), we are investigating the possibility of directed evolution to evolve the translation machinery in laboratory for cell-free system by:
(1) Searching nuclease-resistant hairpin structure (RNA
oligomer fragment) for the stabilization of mRNA-template for
cell-free translation system.
(2) Optimization of 5’-UTR (length versus base composition)
to improve the mRNA-ribosome interaction and thus the regulation of
translation initiation.
This is proposed by introduction of random nucleotide
sequences into sites of interest on mRNA molecules and subsequent
screening of the mRNA pools followed by identification of successful
candidates by protein-fusion method.
Autonomous cell-free evolution of RNA
polymerase and reverse transcriptase
The present scenario about RNA molecules has stimulated our
speculation of RNA-world and that RNA might be an active and star
player of molecular biology- a molecule that contains genetic
information and acts as a biological catalyst. Thus
RNA-directed evolution can provide clues to its possible
participation in the origin of life. Forcefully evolution of a true
RNA replicase as a self-replicating genetic molecule in the in
vitro system using directed molecular evolutionary approach
would provide a window to observe the force of Darwinian selection
in real-time dimension scale. RNA replicase which is exclusively
reserved for viruses (not cellular RNAs) is an RNA-directed RNA
polymerase (RdRP) and can catalyzed the replication of RNA molecules
and even is good enough to copy their own sequence. Thus RNA
replicase would able for setting up an in vitro system to
actually watch how the molecule evolves. However, to get an
evolutionary line for replicase in a selective manner, a RNA
molecule must be evolve in an intramolecular sense in order to boost
its own survival by preferentially replicating itself and not the
other RNAs present in the local environment. In this research, we
wish to attempt and explore the possibility of directed evolution to
evolve a new replicase by combining the potential of self-sustained
sequence replication (3SR) for amplification purposes and
solid-phase RNA-protein fusion for selection purposes. Using this
approach, a RNA candidate which coded functionally active replicase
would be able to amplify immediately and then the amplified product
would be able to catalyze immediately. Therefore, a joint approach
of RNA catalysis shown by RNA-protein fusion followed by RNA
amplification performed by 3SR reinforces the power of directed
molecular evolution for rapid and continuous generation of
functional RNAs and thus may help us to understand the origin of
life scenario based on efficiency and versatility of
self-replicating RNA molecules.
Optimization of synthesis of RNA-protein
fusion
Puromycin-assisted RNA-protein fusion is likely to be more
generally applicable to cell-free protein selection and evolution;
however the selection of maximum library size is limited by the
efficiency of the translation reaction and by the efficiency of
fusion formation on the ribosome. At present, the efficiency of
fusion formation is limited from minimum <1 to maximum ~20% of the
input template, critically depending on the high-quality of
synthetic oligonucleotide containing puromycin at its 3’-end which
is used to make covalent linkage between the mRNA molecules and
their encoded protein product. In this research, we are engineering
and optimizing better construction of oligomer DNA for efficient
preparation of RNA-protein fusion which is also advantageous for
both the stability and handling of larger transcripts. |
