Faculty Profiles - MURAKAMI Hiroshi

Papers - MURAKAMI Hiroshi

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Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method International journal

Naohiro Taniguchi, Hiroshi Murakami

Methods in Molecular Biology Vol. 1498 page： 339 - 347 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer New York

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

DOI： 10.1007/978-1-4939-6472-7_22

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Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

Masuda J, Kawamoto H, Strober W, Takayama E, Mizutani A, Murakami H, Ikawa T, Kitani A, Maeno N, Shigehiro T, Satoh A, Seno A, Arun V, Kasai T, Fuss IJ, Katsura Y, Seno M

Applied biochemistry and biotechnology Vol. 180 ( 8 ) page： 1559 - 1573 2016.12

Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes

Yoshihiko Iwane, Azusa Hitomi, Hiroshi Murakami, Takayuki Katoh, Yuki Goto, Hiroaki Suga

Nature Chemistry Vol. 8 ( 4 ) page： 317 - 325 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNA(SNN) (tRNA(SNN)) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNA(SNN)s with tRNA(SNN)s pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

DOI： 10.1038/nchem.2446

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Ribosomal Synthesis of Peptides with Multiple β-Amino Acids

Tomoshige Fujino, Yuki Goto, Hiroaki Suga, Hiroshi Murakami

Journal of the American Chemical Society Vol. 138 ( 6 ) page： 1962 - 1969 2016.2

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

The compatibility of beta-amino acids with ribosomal translation was studied for decades, but it has been still unclear whether the ribosome can accept various beta-amino acids, and whether the ribosome can introduce multiple beta-amino acids in a peptide. In the present study, by using the Escherichia coli reconstituted cell-free translation system with a reprogramed genetic code, we screened beta-amino acids that give high single incorporation efficiency and used them to synthesize peptides containing multiple beta-amino acids. The experiments of single beta-amino acid incorporation into a peptide revealed that 13 beta-amino acids are compatible with ribosomal translation. Six of the tested beta-amino acids (beta hGly, L-beta hAla, L-beta hPhg, L-beta hMet, and D-beta hPhg) showed high incorporation efficiencies, and seven (L-beta hLeu, L-beta hIle, L-beta hAsn, L-beta hPhe, L-beta hLys, D-beta hAla, and D-beta hLeu) showed moderate incorporation efficiencies; whereas no full-length peptide was produced using other beta-amino acids (L-beta hPro, L-beta hTrp, and L-beta hGlu). Subsequent double-incorporation experiments using beta-amino acids with high single incorporation efficiency revealed that elongation of peptides with successive beta-amino acids is prohibited. Efficiency of the double-incorporation of the beta-amino acids was restored by the insertion of Tyr or Ile between the two beta-amino acids. On the basis of these experiments, we also designed mRNA sequences of peptides, and demonstrated the ribosomal synthesis of peptides containing different types of beta-amino acids at multiple positions.

DOI： 10.1021/jacs.5b12482

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<i>In Vitro</i>Selection Combined with Ribosomal Translation Containing Non-proteinogenic Amino Acids

Tomoshige Fujino, Hiroshi Murakami

The Chemical Record Vol. 16 ( 1 ) page： 365 - 377 2016.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

The potential applications of non-proteinogenic amino acids have increased continuously since the introduction of these molecules into a ribosomal translation system. An increasing number of studies concerning topics, such as the addition of an artificial function to a protein, cellular expression of a protein with an artificial residue, and development of an artificial peptide with a novel function, have been done using these molecules. Here, we describe recent studies that elucidate the compatibility of non-proteinogenic amino acids with ribosomal translation. We also describe the development of a simple and high-speed selection method and its potential application for the creation of a novel functional peptide with non-proteinogenic amino acids. As these studies have expanded the diversity of the artificial peptide library and increased the speed of novel functional peptide selection, they will significantly facilitate the development of new molecules, such as pharmaceutical drug candidates and bioassay probes.

DOI： 10.1002/tcr.201500239

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Incorporation of electrically charged N-alkyl amino acids into ribosomally synthesized peptides via post-translational conversion. Reviewed

Kawakami, T.*; Sasaki, T; Reid, P. C.; Murakami, H.

Chemical Science Vol. 5 page： 887-893 2014.11

ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK–RSK pathway Open Access

Shungo Adachi, Masae Homoto, Rikou Tanaka, Yusaku Hioki, Hiroshi Murakami, Hiroaki Suga, Masaki Matsumoto, Keiichi I. Nakayama, Tomohisa Hatta, Shun-ichiro Iemura, Tohru Natsume

Nucleic Acids Research Vol. 42 ( 15 ) page： 10037 - 10049 2014.9

Language：English Publishing type：Research paper (scientific journal) Publisher：Oxford University Press ({OUP})

<jats:title>Abstract</jats:title>
<jats:p>Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.</jats:p>

DOI： 10.1093/nar/gku652

Open Access

Web of Science

PubMed

Incorporation of electrically charged N-alkyl amino acids into ribosomally synthesized peptides via post-translational conversion

Takashi Kawakami, Toru Sasaki, Patrick C. Reid, Hiroshi Murakami

Chemical Science Vol. 5 ( 3 ) page： 887 - 887 2014

Nonstandard Peptide Expression under the Genetic Code Consisting of Reprogrammed Dual Sense Codons

Yuki Goto, Megumi Iseki, Azusa Hitomi, Hiroshi Murakami, Hiroaki Suga

ACS Chemical Biology Vol. 8 ( 12 ) page： 2630 - 2634 2013.12

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

We here demonstrate a translation system that is governed by a reprogrammed genetic code consisting of "dual sense codons." A dual sense codon assigns two distinct amino acids for initiation and elongation. Because multiple dual sense codons independently function without cross-readings, this system enables the expansion of the repertoire of initiators as well as elongators that can be used simultaneously.

DOI： 10.1021/cb400549p

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Patch cloning method for multiple site-directed and saturation mutagenesis Open Access

Naohiro Taniguchi, Sayumi Nakayama, Takashi Kawakami, Hiroshi Murakami

BMC Biotechnology Vol. 13 ( 1 ) page： 91 2013.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

<jats:title>Abstract</jats:title>
<jats:sec>
<jats:title>Background</jats:title>
<jats:p>Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Results</jats:title>
<jats:p>To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.</jats:p>
</jats:sec>

DOI： 10.1186/1472-6750-13-91

Open Access

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Extensive Reprogramming of the Genetic Code for Genetically Encoded Synthesis of Highly N-Alkylated Polycyclic Peptidomimetics

Takashi Kawakami, Takahiro Ishizawa, Hiroshi Murakami

Journal of the American Chemical Society Vol. 135 ( 33 ) page： 12297 - 12304 2013.8

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Cyclic structures can increase the proteolytic stability and conformational rigidity of peptides, and N-alkylation of the peptide backbone can make peptides more cell-permeable and resistant to proteolysis. Therefore, cyclic N-alkyl amino acids are expected to be useful building blocks to increase simultaneously these pharmacological properties of peptides. In this study, we screened various cyclic N-alkyl amino acids for their ribosomal incorporation into peptides and identified cyclic N-alkyl amino acids that can be efficiently and successively incorporated. We also demonstrated genetic code reprogramming for reassigning 16 NNU codons to 16 different cyclic N-alkyl amino acids with high fidelity to synthesize highly N-alkylated polycyclic peptidomimetics and an mRNA-displayed library of completely N-alkylated polycyclic peptidomimetics by using our recently developed TRAP (transcription/translation coupled with association of puromycin linker) display. In vitro selection from a highly diverse library of such completely N-alkylated polycyclic peptidomimetics could become a powerful means to discover small-molecule ligands such as drug candidates that can be targeted to biomolecules inside living cells.

DOI： 10.1021/ja405044k

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<i>In Vitro</i> Selection of Multiple Libraries Created by Genetic Code Reprogramming To Discover Macrocyclic Peptides That Antagonize VEGFR2 Activity in Living Cells

Takashi Kawakami, Takahiro Ishizawa, Tomoshige Fujino, Patrick C. Reid, Hiroaki Suga, Hiroshi Murakami

ACS Chemical Biology Vol. 8 ( 6 ) page： 1205 - 1214 2013.6

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

We report the in vitro selection of thioether-macrocyclized peptides against vascular endothelial growth factor receptor 2 (VEGFR2) from multiple, highly diverse peptide libraries constructed utilizing genetic code reprogramming. The macrocyclic peptide libraries consisted of combinations of four types of amino acid linkers for cyclization and two types of elongator amino acid compositions, including four backbone-modified non-proteinogenic amino acids. Affinity selection from these libraries, using our recently developed TRAP (Tran scription-translation coupled with Association of Puromycin-linker) display, yielded multiple anti-VEGFR2 macrocydic peptide leads. Further antagonizing activity-based screening of the chemically synthesized lead peptides identified a potent macrocyclic peptide that inhibited VEGF-induced VEGFR2 autophosphorylation, proliferation, and angiogenesis of living vascular endothelial cells. The TRAP display-based selection from multiple, highly diverse peptide libraries followed by activity-based screening of selected peptides is a powerful strategy for discovering biologically active peptides targeted to various biomolecules.

DOI： 10.1021/cb300697h

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TRAP Display: A High-Speed Selection Method for the Generation of Functional Polypeptides

Takahiro Ishizawa, Takashi Kawakami, Patrick C. Reid, Hiroshi Murakami

Journal of the American Chemical Society Vol. 135 ( 14 ) page： 5433 - 5440 2013.4

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

Here, we describe a novel method that enables highspeed in vitro selection of functional peptides, peptidomimetics, and proteins via a simple procedure. We first developed a new cell-free translation system, the TRAP system (transcription translation coupled with association of Euromycin linker), which automatically produces a polypeptide library through a series of sequential reactions: transcription, association of puromycin-DNA linker, translation, and conjugation between the nascent polypeptide and puromycin-DNA linker. We then applied the TRAP system for the selection of macrocyclic peptides against human serum albumin. Six rounds of selection using TRAP display were performed in approximately 14 h, yielding macrocyclic peptides with nanomolar affinity to their target protein. Because TRAP display enables high-speed selection of functional polypeptides, it will facilitate the generation of various polypeptides that are useful for biological and therapeutic applications.

DOI： 10.1021/ja312579u

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Reevaluation of the <scp>d</scp>-Amino Acid Compatibility with the Elongation Event in Translation

Tomoshige Fujino, Yuki Goto, Hiroaki Suga, Hiroshi Murakami

Journal of the American Chemical Society Vol. 135 ( 5 ) page： 1830 - 1837 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society ({ACS})

The compatibility of D-amino acids with peptide elongation during translation has been examined in several studies. However, some of the studies have reported that D-amino acids are incompatible with translation, whereas others have reported that D-amino acids are incorporated into polypeptides. Here, we have reevaluated the incorporation of a series of D-amino acids into the nascent chain of short peptides with a reprogrammed genetic code by using the flexible in vitro translation (FIT) system. The FIT system enables the compatibility of each D-amino acid with elongation to be assessed quantitatively in the absence of potential competitors. The incorporation efficiencies were determined by Tricine-SDS-PAGE and the full-length peptide was detected by MALDI-TOF-MS. The D-amino acids were categorized into three groups based on their incorporation efficiencies relative to the corresponding L-amino acid. The D-isomers in group I showed efficiencies of 40% or higher (Ala, Ser, Cys, Met, Thr, His, Phe, and Tyr), and those in group H showed efficiencies of 10-40% (Asn, Gln, Val, and Leu). The D-amino acids in group III produced truncated peptides or no detectable full-length peptides (Arg, Lys, Asp, Glu, Ile, Trp, and Pro). When group I D-amino acids were used consecutively or were alternated with L-amino acids, this completely inhibited their elongation. However, when two or three L-amino acids were inserted between the D-amino acids, the double-incorporation efficiency was restored. Our results quantitatively reveal the compatibility of D-amino acids with peptide elongation and raise new questions about the mechanism of D-amino acid selection and incorporation by the ribosome.

DOI： 10.1021/ja309570x

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Patch cloning method for multiple site-directed and saturation mutagenesis Reviewed Open Access

Taniguchi, N; Nakayama, S; Kawakami, T.; Murakami, H.

BMC Biotechnology page： 13:91 2013

Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics. Reviewed

Kawakami, T.; Ishizawa, T.; Murakami, H

Journal of the American Chemical Society Vol. 135 page： 12297-304 2013

Artificially created nucleic acids and peptides/proteins in chemical biology.

Kuwahara M, Li Y, Rozners E, Murakami H

Journal of nucleic acids Vol. 2013 page： 219263 2013

Genetically Encoded Libraries of Nonstandard Peptides

Takashi Kawakami, Hiroshi Murakami

Journal of Nucleic Acids Vol. 2012 page： 1 - 15 2012

Language：English Publishing type：Research paper (scientific journal) Publisher：Hindawi Limited

<jats:p>The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.</jats:p>

DOI： 10.1155/2012/713510

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Diverse backbone-cyclized peptides via codon reprogramming

Takashi Kawakami, Atsushi Ohta, Masaki Ohuchi, Hiroshi Ashigai, Hiroshi Murakami, Hiroaki Suga

Nature Chemical Biology Vol. 5 ( 12 ) page： 888 - 890 2009.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

We report a methodology for the ribosomal synthesis of backbone-cyclized peptides involving genetic code reprogramming to introduce one or more nonproteinogenic amino acids. Expression of linear peptides bearing a cysteine-proline dipeptide sequence followed by glycolic acid results in self-rearrangement to a C-terminal diketopiperadine-thioester, which non-enzymatically generates a cyclized peptide. We demonstrate the ribosomal synthesis of several naturally occurring backbone-cyclized peptides and a library based on a bicyclic scaffold, and we identify bioactive sequences by screening and deconvolution.

DOI： 10.1038/nchembio.259

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A flexizyme that selectively charges amino acids activated by a water-friendly leaving group

Nobuyoshi Niwa, Yusuke Yamagishi, Hiroshi Murakami, Hiroaki Suga

Bioorganic & Medicinal Chemistry Letters Vol. 19 ( 14 ) page： 3892 - 3894 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier {BV}

We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water solubility. Although the previously reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexizyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selectivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2009.03.114

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