mRNA-protein fusion technology

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 days 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 mRNA-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 (see Fig. below). 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.

We have introduced next generation of this technology, called solid-phase mRNA-protein fusion, and extending its potential to develop “Protein biochips” for large-scale and high-throughput proteomics. Protein biochip usually refers to the protein microarray, which has an orderly arrangement of several thousands of different proteins.
Since disease-causing changes seen at the protein level and may not occur at the DNA/RNA level, development of novel protein biochip systems are very promising to add “functional flesh” to the “bare bones” of encoding genes and thus leading the way to rapidly profile the entire proteome. However, compared with DNA chips, protein chips provide more challenge due to the complexity and inherent difficulties. Such as: like DNA, it is not possible to amplify proteins and thus to detect very tiny amounts; it is not easy to attach the proteins to chips;
it is not easy to maintain their integrity and stability as proteins tend to adsorb non-specifically to surfaces and leading the possibility of denaturation and loss-of-function. In addition, the display of proteins in microarray format is a problem for which there is no general solution yet. To foster these challenges, we are focusing on engineering and converging microarray reactors and solid-phase RNA-protein fusion techniques to create high-throughput functional protein arrays directly from encoding genes.
This enables parallel analyses of high-density protein microarray on the scale of a single-molecule while displaying both the genetic (encoding DNA/RNA) and the functional (encoded protein) information contents on-a-chip. To build the platform, we have developed a microfabricated chip using photolithography which is comprising of uniformly distributed sub-picoliter scale reactors of 6µm in diameter (see Fig. below).

References:

• Manish Biyani, Terutsune Osawa, Naoto Nemoto and Takanori Ichiki (2009) DNA-linked protein array:
      a single molecule-derived self-addressable array platform for next generation proteomics. Proteomics     (submitted).
• Junichi Yamaguchi, Manish Biyani, Yuzuru Husimi, Takashi Funatsu and Naoto Nemoto (2009) cDNA
      display: A novel method for rapid stabilization of mRNA-protein fusion and its application to the screening
      of functional proteins. Nucleic Acids Research(submitted).
• Manish Biyani, Terutsune Osawa, Naoto Nemoto and Takanori Ichiki (2008) One-to-one gene-encoded
      functional protein microarray. In: Proc of 12th International Conference on Miniaturized Systems for
      Chemistry and Life Sciences. MicroTAS, San Diego, USA, vol.2, p.p.1781-1783.
• Manish Biyani, Naoto Nemoto and Yuzuru Husimi (2006) Solid-phase translation and RNA-protein
      fusion: a novel approach for folding quality control and direct immobilization of protein using anchored
      mRNA. Nucleic Acids Research 34, e140.
• Manish Biyani, Naoto Nemoto, Yosuke Hosoi and Takanori Ichiki (2006) RNA-to-Protein Chip: an
      integrated microarray platform to bridge genomics and proteomics. In: Proc. of 10th International
      Conference on Miniaturized Systems for Chemistry and Life Sciences. MicroTAS, Tokyo, Japan, vol.1,
      p.p.209-211
• Yosuke Hosoi, Manish Biyani, Naoto Nemoto, Takanori Akagi and Takanori Ichiki (2005)
      High-throughput screening of mutant biomolecules using mRNA display and microreactor array chips. In:
      Proc. of International Conference on Solid State Device and Materials, SSDM, Kobe, Japan, pp.452-453.
• Yosuke Hosoi, Manish Biyani, Naoto Nemoto and Takanori Ichiki (2005) High-Throughput Screening of
      mutant AKR enzymes using mRNA display and novel microreactor array chips. In: Proc. of 9th
      International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS, Boston,
      USA, vol.1, p.p. 323-325.