FY2020 Annual Report

Nucleic Acid Chemistry and Engineering Unit
Professor Yohei Yokobayashi

 

Abstract

FY2020 started with COVID-19 suspension of laboratory research activities. Reseach efforts continued in RNA engineering of cell-free and mammalian systems while new projects in microbial toxins that target nucleic acids have been initiated. A new collaborative research with an industrial partner has also started with dedicated funding. Finally, the first graduate student from the unit was awared Ph.D. near the end of the fiscal year. 

 

1. Staff

  • Dr. Keisuke Fukunaga, Researcher
  • Dr. Samuel Hauf, Researcher
  • Dr. Narae Kim, Researcher
  • Dr. Yoko Nomura, Science and Technology Associate
  • Dr. Vyankat Sontakke, Researcher
  • Kamila Mustafina, Graduate Student
  • Takeshi Tabuchi, Graduate Student
  • Rachapun (Gear) Rotrattanadumrong, Graduate Student
  • Nao Miyahira, Technical Staff
  • Mayumi Suzuki, Technical Staff
  • Hitomi Shinzato, Research Unit Administrator
  • Yayoi Maehara, Research Assistant

(As of 3/31/2021)

2. Collaborations

2.1 Applications of aptamers for biosensors

  • Description: Using DNA aptamers to sense proteins using bionanosensors 

  • Type of collaboration: Joint research

  • Researchers:

    • Professor Mukhles Sowwan, OIST (Nanoparticles by Design Unit)

2.2 Collaboration with an industrial partner

  • Undisclosed collaboration with an industrial partner.

 

3. Activities and Findings

3.1 Riboswitches in Cell-Free Systems and Artificial Cells 

Artificial cell models composed of lipid vesicles (liposomes) encapsulating an in vitro (cell-free) transcription-translation system (IVTT) are emerging as a part of the efforts to synthesize cells from bottom up. Researchers have constructed artificial cells that mimic various biological properties such as DNA replication, cell-cell communication, and cell division. Last year, we reported histamine responsive riboswitches in cell-free system and artificial cells in collaboration with Prof. Matsuura at Osaka University (Dwidar et al. 2019). In this work, we recognized challenges in designing riboswitches in cell-free systems, for example, the lack of high-throughput screening methods.

This fiscal year, we focused on development of histamine and other chemically responsive riboswitches in cell-free systems. In particular, we developed a high-throughput screening strategy for identifying riboswitches in cell-free systems from a large pool of candidates. Validation of the method and characterization of the novel riboswitches are ongoing.

 

3.2 Minimization of an RNA Ligase Ribozyme

We used the deep sequencing-based assay to analyze and minimize an important class of laboratory-evolved ribozymes, an RNA ligase ribozyme that catalyzes the template-directed ligation of an RNA fragment’s 3’-OH group and the 5’-triphosphorylated RNA fragment (Fig. 1) in FY2019. This fiscal year, we have explored even smaller RNA sequences capable of catalyzing the same reaction. We identified a number of promising candidates from in vitro selection and sequencing, and these mini-ribozymes will be further characterized in the next fiscal year.

Fig. 1 Minimized RNA ligase ribozyme 4d394. From Nomura & Yokobayashi 2019.


 

3.3 Novel Ribozyme Scaffold for Mammalian Riboswitches 

We and others have been developing synthetic riboswitches that allow chemical regulation of gene expression in mammalian cells. These riboswitches are typically engineered using self-cleaving ribozymes and an RNA aptamer that specifically bind a small molecule. Ribozymes that have been used to engineer mammalian riboswitches have been limited to only three classes (hammerhead, HDV, and twister). In this project we have focused on developing a new self-cleaving ribozyme scaffold for developing riboswitches. Addition of a new ribozyme scaffold broadens the design options for engineering riboswitches that respond to new chemical signals and genetic contexts. This fiscal year, we collected comprehensive data on the novel ribozyme scaffold and riboswitches in mammalian cells for a publication in the coming fiscal year.

3.4 Development of Novel VSV Vector and Applications 

Vesicular stomatitis virus (VSV) is a unique virus with potential clinical applications. VSV is a nonsegmented, negative-sense, cytoplasmic RNA virus that replicates without DNA intermediates; therefore, there is no risk of genomic integration. Furthermore, VSV is virtually nonpathogenic to humans and can stably (>weeks) and strongly express exogenous genes. VSV vectors have been investigated for applications as oncolytic viruses and vaccine vectors.

We reported an engineered VSV vector that can be chemically regulated by embedded riboswitches in FY19 (Takahashi & Yokobayashi 2020). This fiscal year, we continued to develop improved vectors for biological and biomedical applications. Details are not disclosable due to a pending patent application.

 

4. Publications

4.1 Journals

  1. Danielson E, Sontakke VA, Porkovich AJ, Wang Z, Kumar P, Ziadi Z, Yokobayashi Y, Sowwan M. Graphene Based Field-Effect Transistor Biosensors Functionalized Using Gas-Phase Synthesized Gold Nanoparticles. Sens Actuators B Chem 2020; 320, 128432.
  2. Yokobayashi Y. High-Throughput Analysis and Engineering of Ribozymes and Deoxyribozymes by Sequencing. Acc Chem Res 2020; 53, 2903–2912.
  3. 横林洋平. セルフリーシステムにおける人工リボスイッチの構築. 生物工学会誌　2020; 98, 655-658. (A review in Japanese, Yohei Yokobayashi, Seibutsu Kogaku Kaishi)
  4. Kim N. pH variation impacts molecular pathways associated with somatic cell reprogramming and differentiation of pluripotent stem cells. Reprod Med Biol 2020; 20: 20–26.

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

  1. Yokobayashi, Y. Engineering Synthetic Riboswitches in Cell-Free Systems (セルフリーシステムにおける人工リボスイッチの構築), SBJ Web Symposium 2020 (The Society for Biotechnology, Japan 生物工学Webシンポジウム2020), Japan, Sep 2-3 (2020), Invited talk.
  2. Fukunaga, K., Yokobayashi, Y. Selection of Orthogonal RNA-RNA Binding Protein Pairs via Library vs Library Coevolution (ライブラリー vs. ライブラリーの共進化実験を通じた 直交性RNA-RNA結合タンパク質ペアの探索),  14th Bio-Related Chemistry Symposium (バイオ関連化学シンポジウム2020), Japan, Sep 7-8 (2020). Poster.
  3. Mustafina, K., Fukunaga, K., Yokobayashi, Y. Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme, Virtual International Mammalian Synthetic Biology Workshop (mSBW), Dec 7-8 (2020). Poster.
  4. Fukunaga, K., Yokobayashi, Y. Selection of Orthogonal RNA-RNA Binding Protein Pairs via Library vs Library Coevolution (ライブラリー vs. ライブラリーの共進化実験を通じた 直交性RNA-RNA結合タンパク質ペアの探索),  The 101st CSJ Annual Meeting (日本化学会第101春季年会), Japan, Mar 19-22 (2021). Poster.

5. Intellectual Property Rights and Other Specific Achievements

Patent: AN RNA APTAMER THAT SPECIFICALLY BINDS HISTAMINE (WO 2020/171070)

6. Meetings and Events

Nothing to report

7. Other

Research Intern

Shivang Joshi, Jan-Jul 2020

Degree Awarded

Kamila Mustafina, Ph.D. Feb 28, 2020.

Outreach activities

Yoko Nomura (November 2020): contributed an article (in Japanese) written for the general public in "Green Age" magazine published by Japan Greenery Research and Development Center. 

Takeshi Tabuchi (August 2, 2020): taught a 2-hour online lecture for master's students (“RNA engineering: Aptamers, Riboswitches, and Ribozymes.”) in Universidad Nacional Mayor de San Marcos (Peru).

Takeshi Tabuchi (February 26, 2021): Public talk (online) sponsored by Junkudo Bookstore (Naha).

External funding and support

Yohei Yokobayashi: Undisclosed industry partner (new, PI), KAKENHI Kiban B 19H02855 (continuing, PI), KAKENHI Challenging Research Exploratory 18K19944 (continuing, PI)

Keisuke Fukunaga: KAKENHI Young Researcher (continuing, PI) 19K15701

Narae Kim: KAKENHI Young Resercher (new, PI) 20K15669

Kei Takahashi: KAKENHI Young Researcher (continuing, PI) 19K15988

Samuel Hauf: Walter Benjamin Fellowship (from Deutsche Forschungsgemeinschaft, new)