Genetic code reprogramming: A new emerging technology for the discovery of a new class of > Seminar

Genetic code reprogramming: An emerging technology for the discovery of a new class of peptidic drugs

Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

The genetic code is the law of translation, where genetic information encoded in RNA is translated to amino acid sequence. The code consists of tri-nucleotide sequences, so-called codons, assigning to particular amino acids. In cells or in ordinary cell-free translation systems originating from prokaryotes or eukaryotes, the usage of amino acids is generally restricted to 20 proteinogenic (standard) kinds, and thus the expressed peptides are composed of only such monomers. However, we recently devised a new means to reprogram the genetic code, which allows us to express “non-standard” peptides containing multiple non-proteinogenic amino acids.

To execute the genetic code reprogramming, we developed an artificial RNA enzyme (ribozyme), referred to as “flexizyme” capable of aminoacylating tRNAs. The most notable feature of flexizyme is its versatility; it is able to charge virtually any α-amino acids onto tRNAs bearing various anticodons. Moreover, the flexizyme also charges α-amino acids with non-proteinogenic sidechain, D-α-amino acids, N-alkyl-amino acids, β-amino acids, and even hydroxy acids onto tRNA.1-3 Thus, the reassignment of such non-proteinogenic amino acids to We also integrated this unique enzyme system with a specially reconstituted E. coli cell-free transcription-translation coupled system, from which certain amino acids (occasionally their cognate aminoacyl α-tRNA synthetases) were withdrawn to vacant the corresponding codons, referred to as wPURE system (w stands withdrawn, and PURE stands Protein translation Using Recombinant Elements). By the integration of these two systems, any desired amino and hydroxy acids can be reassigned to the vacant codons, and we have recently showed that a wide variety of non-standard peptides can be expressed from mRNAs under the reprogrammed genetic code in the wPURE system.

Since the flexizyme system was developed by my research group, we know everything about its capabilities. In last two years, we made major efforts to develop new methodologies for the ribosomal expression of cyclic non-standard peptides closed by a non-reducible bond, allowing us to prepare even their libraries now. We therefore are on a firm ground that enables us to extensively explore the sequence space of non-standard peptides for biological activities. Particularly, we succeeded in coupling the cyclic non-standard peptides with the mRNA display technique and also developed a unique rapid screening technique for functional assays.4-13 We referred to the system involving these techniques as RaPID (Random Peptide Integrated Discovery) system. The seminar will summarize our recent progress in accelerating the discovery of natural product-like cyclic peptides that agonize or antagonize biological functions in cells and potentially in human as new therapeutic agents. Our ultimate goal is to develop a new class of peptidic drugs that potentially fulfill the gap between small organic molecules and biologics, and thus open a new frontier in pharmaceutical research.

Acknowledgement:
This work was supported by grants of Japan Society for the promotion of Science Grants-in-Aid for Scientific Research (S) (16101007) and a research and development projects of the Industrial Science and Technology Program in the New Energy and Industrial Technology Development Organization (NEDO) to H.S..

References

"The fexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides" H. Murakami, A. Ohta, H. Ashigai, H. Suga Nature Methods 3, 357-359 (2006).
"The flexizyme system: A highly flexible tRNA aminoacyation tool for the translation apparatus" M. Ohuchi, H. Murakami, H. Suga Current Opinion in Chemical Biology 11, 135-144 (2007). # "Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids" T. Kawakami, H. Murakami, H. Suga Journal of American Chemical Society 130, 16861-16863 (2008).
"Strcutural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme" H. Xiao, H. Murakami, H. Suga, A. R. Ferre-D'Amare Nature 454, 358-361 (2008).
"Combinatorial lysine modifications of histone H3 tails under the reprogrammed genetic code suggest crosstalk between epigentic markers upon HP1 chromo domain binding" T.-J. Kang, S. Yuzawa, H. Suga Chemistry & Biology 15, 1166-1174 (2008).
"Polymerization of ?α-hydroxy acids by ribosomes" A. Ohta, H. Murakami, H. Suga ChemBioChem in press (2008).
"Initiating translation with D-amino acids" Y. Goto, H. Murakami, H. Suga RNA 14, 1399-1410 (2008).
"Ribosomal synthesis of bicyclic peptides with two orthogonal inter-sidechain reactions" Y. Sako, J. Morimoto, H. Murakami, H. Suga Journal of American Chemical Society 130, 7932-7934 (2008).
"Ribosomal synthesis of nonstandard peptides" T.-J. Kang, H. Suga Biochemistry and Cell Biology 86, 92-99 (2008).
"Synthesis of biopolymers using genetic code reprogramming" A. Ohta, Y. Yamagishi, H. Suga Current Opinion in Chemical Biology 12, 159-167 (2008).
"Ribosomal synthesis of peptidase-resistant peptides closed by a non-reducible inter-sidechain bond" Y. Sako, Y. Goto, H. Murakami, H. Suga ACS Chemical Biology 3, 241-249 (2008).
"Reprogramming the initiation event in translation for the synthesis of physiologically stable cyclic peptides" Y. Goto, A. Ohta, Y. Sako, Y. Yamagishi, H. Murakami, H. Suga ACS Chemical Biology 3, 120-129 (2008).
"Messenger RNA-directed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides" T. Kawakami, H. Murakami, H. Suga Chemistry & Biology 15, 32-42 (2008).
"Synthesis of polyester by means of genetic code reprogramming" A. Ohta, H. Murakami, E. Higashimura, H. Suga Chemistry & Biology 14, 1315-1322 (2007).

Brief CV
Bachelor of Engineering (1986), School of Engineering, University of Okayama (Japan).
Master of Engineering (1989), School of Engineering, Department of Advanced Chemistry, University of Okayama (Japan).
Ph.D. (1994), Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA.
Post-doctoral Fellow (1994-1997), Harvard Medical School/Massachusetts General Hospital, Boston, MA.
Assistant Professor (1997-2002) Dep. of Chemistry, University at Buffalo (SUNY)
Associate Professor (2002-2003) Dep. Of Chemistry, University at Buffalo (SUNY)
Associate Professor (2003-2005) Research Center for Advanced Science and Technology, University of Tokyo
Professor (2005-present) Research Center for Advanced Science and Technology, University of Tokyo