Development of a new method for chemical synthesis of genomic DNAs

• Project/Area Number: 24550184
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: AKIHIRO Ohkubo 東京工業大学, 生命理工学研究科, 准教授 (60376960)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2014: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2012: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
• Keywords: DNA合成 / ホスホロアミダイト / ポーラスガラス / 核酸合成 / DNA / ゲノムDNA / 固相合成

Outline of Final Research Achievements

The final goal of this study is development of a high-throughput method for chemical synthesis of the genomic DNAs of mycoplasma and cyanobacteria. In this research, ‘improvement of chain elongation efficiency’, ‘simplification of purification of oligonucleotides’, and ‘development of a flow-system for synthesis of long oligonucleotides’ was carried out. Specifically, l the 30-mer DNA oligomers was synthesized by using the condensation reaction on the activator-contained solid phase carrier and a capping reaction with diphenylphosphonate. We also prepared a filter-type porous glass carrier for the flow system of DNA synthesis and examined the reactivity on the support in detail.

Research Products

• [Journal Article] Properties of 5- and/or 2-modified 2 '-O-cyanoethyl uridine residue: 2 '-O-cyanoethyl-5-propynyl-2-thiouridine as an efficient duplex stabilizing component (2014)
  • Author(s): Masaki Y, Miyasaka R, Hirai K, Kanamori T, Tsunoda H, Ohkubo A, Seio K, Sekine M
  • Journal Title: ORGANIC & BIOMOLECULAR CHEMISTRY Volume: 12 Pages: 1157-1162
  • DOI: 10.1039/c3ob41983e
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A new modified cytosine base capable of base pairing with guanine using four hydrogen bonds (2014)
  • Author(s): Yamada K, Masaki Y, Tsunoda H, Ohkubo A, Seio K, Sekine M.
  • Journal Title: Org Biomol Chem. Volume: 12 Pages: 2255-2262
  • DOI: 10.1039/c3ob42420k
  • Peer Reviewed
• [Journal Article] Synthesis and properties of oligonucleotides modified with 2´-O-(2-carboxyethyl)nucleotides and their carbamoyl derivatives (2014)
  • Author(s): Yamada T, Masaki Y, Okaniwa N, Kanamori T, Ohkubo A, Tsunoda H, Seio K, Sekine M
  • Journal Title: Organic & Biomolecular Chemistry Volume: 12 Pages: 6457-6464
  • DOI: 10.1039/c4ob01260g
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] 61. Base recognition of gap sites in DNA-DNA and DNA-RNA duplexes by short oligonucleotides (2013)
  • Author(s): Yamada K, Ohkubo A, Esaka Y, Kanamori T, Masaki Y, Seio K, Sekine M.
  • Journal Title: Bioorg. Med. Chem. Lett. Volume: 23 Pages: 3448-3451
  • DOI: 10.1016/j.bmcl.2013.03.054
  • Peer Reviewed
• [Journal Article] 62. Fluorescent properties of oligonucleotides doubly modified with an indole-fused cytosine analog and 2-aminopurine. (2013)
  • Author(s): Seio, K.; Kanamori, T.; Tokugawa, M.; Ohzeki, H.; Masaki, Y.; Tsunoda, H.; Ohkubo, A.; Sekine, M.
  • Journal Title: Bioorg. Med. Chem. Volume: 23 Pages: 3197-3201
  • DOI: 10.1016/j.bmc.2013.03.034
  • Peer Reviewed
• [Journal Article] Chemical synthesis of U1 snRNA derivatives. (2013)
  • Author(s): Ohkubo, A.; Kondo, Y.; Suzuki, M.;Kobayashi, H.;Kanamori, T.; Masaki, Y.; Seio, K.; Nagai, K.; Sekine, M
  • Journal Title: Org. Lett. Volume: 15 Pages: 4386-4389
  • DOI: 10.1021/ol401917r
  • Peer Reviewed
• [Journal Article] 64. Modified oligodeoxynucleotide primers for reverse-transcription of target RNAs that can discriminate among length variants at the 3′-terminus. (2013)
  • Author(s): Iijima, Y.; Kojima, S.; Kodama, E.; Kurohagi, S.; Kanamori, T.; Masaki, Y.; Ohkubo, A.; Sekine, M.; Seio, K.
  • Journal Title: Organic & Biomolecular Chemistry Volume: 11 Pages: 8276-8282
  • DOI: 10.1039/c3ob41901k
  • Peer Reviewed
• [Journal Article] 65. Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region. (2013)
  • Author(s): Seio, K.; Tokugawa, M.; Tsunoda, H.; Ohkubo, A.; Arisaka, F.; Sekine, M
  • Journal Title: Bioorg Med Chem Lett. Volume: 23 Pages: 6822-4
  • DOI: 10.1016/j.bmcl.2013.10.012
  • Peer Reviewed
• [Journal Article] Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups (2013)
  • Author(s): Yamada K, Hattori Y, Inde T, Kanamori T, Ohkubo A, Seio K, Sekine M.
  • Journal Title: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Volume: 23 Pages: 776-778
  • DOI: 10.1016/j.bmcl.2012.11.079
  • Peer Reviewed
• [Journal Article] Prediction of the stability of modified RNA duplexes based on deformability analysis: oligoribonucleotide derivatives modified with 2'-O-cyanoethyl-5-propynyl-2-thiouridine as a promising component (2012)
  • Author(s): Masaki, Y.; Miyasaka, R.;Hirai, K.;Tsunoda, H.; Ohkubo, A.;Seio, K.;Sekine, M
  • Journal Title: CHEMICAL COMMUNICATIONS Volume: 48 Pages: 7313-7315
  • DOI: 10.1039/c2cc33409g
  • Peer Reviewed
• [Journal Article] Synthesis and properties of cationic 2'-O-[N-(4-aminobutyl) modified oligonucleotides (2012)
  • Author(s): Seio, K.; Tokugawa, M.; Kanamori, T.; Tsunoda, H.; Ohkubo, A.; Sekine, M.
  • Journal Title: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Volume: 22 Pages: 2470-2473
  • DOI: 10.1016/j.bmcl.2012.02.012
  • Peer Reviewed
• [Journal Article] Formation of new base pairs between inosine and 5-methyl-2-thiocytidine derivatives. (2012)
  • Author(s): Ohkubo, A.; Nishino, Y.; Ito, Y.; Tsunoda, H.; Seio, K.; Sekine
  • Journal Title: Org. Biomol. Chem. Volume: 10 Pages: 10-13
  • DOI: 10.1039/c2ob06641f
  • Peer Reviewed
• [Journal Article] Development of an efficient method for phosphorodiamidate bond formation by using inorganic salts. (2012)
  • Author(s): T. Harakawa, H. Tsunoda, A. Ohkubo, K. Seio, and M. Sekine,
  • Journal Title: Bioorg. Med. Chem. Lett. Volume: 22 Pages: 1445-1447
  • DOI: 10.1016/j.bmcl.2011.12.021
  • Peer Reviewed
• [Journal Article] Synthesis of 5'-terminal capped oligonucleotide using O-N phosphoryl migration of phosphoramidite derivatives. (2012)
  • Author(s): Ohkubo, A.;Tago, N.;Nishino, Y.;Yamada, K.; Tsunoda, H.:Seio, K.:Sekine, M
  • Journal Title: Org. Lett. Volume: 14 Pages: 10-13
  • DOI: 10.1021/ol2026075
  • Peer Reviewed
• [Presentation] Synthesis of oligodeoxynucleotides incorporating conformational restriction at the 5’-terminus and their application to discrimination of RNA length variants (2013)
  • Author(s): Yoshihiro Iijima, Sayako Kurohagi, Erika Kodama, Shun Kojima, Takashi Kanamori, Yoshiaki Masaki, Akihiro Ohkubo, Mitsuo Sekine, Kohji Seio
  • Organizer: The 40th international symposium on nucleic acids chemistry
  • Place of Presentation: Kanagawa University (Yokohama)
• [Presentation] Chemical synthesis of U1 snRNA derivatives (2013)
  • Author(s): Haruki Kobayashi, Akihiro Ohkubo, Makoto Suzuki, Takashi Kanamori, Yoshiaki Masaki, Kohji Seio, Mitsuo Sekine, Hideya Yuasa
  • Organizer: The 40th international symposium on nucleic acids chemistry
  • Place of Presentation: Kanagawa University (Yokohama)
• [Presentation] Development of fluorescent turn-on sensor for triplex formation and its application to nucleosides and oligonucleotides sensing (2012)
  • Author(s): Takashi Kanamori, Hiroki Ohzeki, Yuki Oda, Akihiro Ohkubo, Mitsuo Sekine, Kohji Seio
  • Organizer: The 39th International Symposium onNucleic Acids Chemistry
  • Place of Presentation: Nagoya, Japan
• [Presentation] Synthesis and properties of new triplex forming oligonucleotides capable of recognizing TA and CG base pairs in parallel orientation (2012)
  • Author(s): Kenji Yamada, Yu Ito, Kiichi Yoshimura, Takashi Kanamori, Akihiro Ohkubo, Kohji Seio, Mitsuo Sekine
  • Organizer: The 39th International Symposium onNucleic Acids Chemistry
  • Place of Presentation: Nagoya, Japan
• [Remarks] 湯浅・大窪研究室
  • URL: http://www.yuasa-lab.bio.titech.ac.jp/
• [Remarks] 湯浅・大窪研究室
  • URL: http://www.yuasa-lab.bio.titech.ac.jp/