cDNA display technology（patent pending）
Huge library of sequences diversity（1013-14）
- Single domain antibody（VHH）
- Alpaca naïve VHH library
- PharmaLogicalTMVHH library（It will be implemented in 2021）
- Cyclic peptide: 8~13 randomized amino acids library
High through put screening method (patent pending)
- SDNC system: In vitro Selection by Differencing operation using NGS and Cell-Sorter with cDNA display
- NGS analyzing software（Joint research results with Mitsui Knowledge Industry Co., Ltd.）
- Several types of transmembrane proteins (GPCR, transporter, channel proteins) are constructed on the Nanodisc or liposome.
※PharmaLogical is applied for trade mark registration in Japan
Our large and high quality library
EME VHH antibody library/EME cyclic peptide library
The diversity of library：１０１3−１４
EME VHH library
(M.W 12-15 kDa, PDB: 1l3V)
Advantages of VHH
・High structural stability (at low pH or high temperature) / protease resistance
・Preparation with bacterial expression system
・Versatility of antigen binding interface (convex, concave, flat and penetrative)
・Easy of modifications to multivalent
VHH antibody shows the same degree (nM order) of antigen affinity as lgG.
Comparison of characteristics of lgG antibody and VHH antibody
|Conventional antibody (lgG)（IgG）||VHH antibody|
|Production by microorganisms||difficult||Easy|
|Refolding from denaturing condition||Impossible||Easy|
|Thermal stability||Low||Very high|
|Acquiring neutralizing antibody||Difficult||Easy|
EME cyclic peptide library
EME cyclic peptide
Formation of cyclic peptides with the chemical linker.
Libraries with various characteristics
- The hydrophobic/hydrophilic amino acids rich library
- 8 to 13 amino acids (equally randomized)
Our unique linker
Structure of photo-crosslink puromycin linker（cnvK linker）
The new puromycin linker containing 3-cyanovinylcarbazole nucleoside (cnvK)
For cDNA display method, the puromycin linker is the key technology of genotype-phenotype linking strategy. As this puromycin linker, we use our unique puromycin linker which is containing 3-cyanovinylcarbazole nucleoside (cnvK), and it is called cnvK linker.
Firstly, the cnvK linker is hybridized with specific region of mRNA. After that, The cnvK linker is hybridized to the mRNA and photo-cross-linked by UV irradiation thenthe mRNA-cnvK linker cross-linked product is formed. These steps can largely save the time for the preparation of cDNA display molecules. The cnvK-Pu-linker is hybridized to the mRNA and photo-cross-linked by UV irradiation.
The advantages of cnvK linker are as follows;
- cnvK linker is able to be ligated with mRNA by photo-cross linkage instead of conventional enzymatic ligation. [Save the reaction time (enzyme: approximately 1hr ⇒ photo-cross linkage: few minutes)]
- Using enzyme for the concatenate cause contamination of samples. However, using cnvK linker doesn’t need application of enzyme then reduce the contamination. Therefore, using cnvK linker can improve the reaction rate of cDNA display molecules because of reduction of contamination.
- cnvK is useable for both in-vitro selection experiment and evaluation of binding affinity.
The method of genotype-phenotype linking strategy with puromycin linker
After the preparation of the mRNA-cnvK linker, the mRNA-cnvK linker isadded to cell free translation reaction mixture . Ribosome start translation from 5’ ends side of mRNA , and polypeptides are synthesized. Because ribosome doesn’t translate the sequence of cnvK linker, ribosome stops translation when ribosome reaches the sequence of cnvK linker. At the same time, puromycin (aminoacyl tRNA analog) in the cnvK linker is integrated into ribosome. When the cnvK linker is integrated into ribosome, the peptide transfer reaction is occurred. Because of the peptide transfer reaction, the C-termini of synthesized polypeptide chain and puromycin linker is connected with covalent bond. Therefore, this synthesized compound is mRNA-cnvK linker polypeptide.
In EME, we use our unique linker “photo-cross linkage puromycin linker (cnvK linker)” and “cDNA display synthesizer” it allows the auto preparation of large diversity (1013-14) of cDNA library.
Schematic illustration of in vitro screening of target binding VHHs or peptide aptamers with cDNA display
The advantages of cDNA display method technology
The preparation of cDNA display molecules enables to be performed in the test tube.
Some display methods are not able to display proteins because some proteins have cytotoxicity and hard to display with cells or microbes such as cell surface display and phage display.
However, cDNA display method enables to display proteins which have cytotoxicity because cDNA display doesn’t require any cells or microbes for translation.
Also, the diversity size of cDNA library is up to approximately 1014 which is much larger than conventional library.
cDNA display method is similar to other types of cell free translation systems such as ribosome display method and mRNA display method. Ribosome display molecule is that ribosome works for the conjugation of mRNA with peptide and protein which were translated by cell free translation system. mRNA display method is the method that puromycin works for the conjugation of mRNA with peptide and protein which were translated by cell free translation system. Comparing these two-display method and cDNA display method, these two display methods have critical disadvantages. For instance, ribosome display method racks the stability because ribosome is easily to be removed from mRNA due to some experimental conditions. For example, high temperature causes the removal of ribosome on the mRNA and it leads to the separation of protein from mRNA. Also, the differences between mRNA display method and cDNA display method are where puromycin is conjugated. For mRNA display method, puromycin is conjugated on the mRNA strand. Thus, enzyme, high temperature, and pH(alkaline) cause the elimination of puromycin from the strand due to the degradation of RNA . For cDNA display method, the puromycin is conjugated on the cDNA strand. Thus, cDNA display method is much stable than ribosome display method and mRNA display method.
Molecular design by cDNA display – From mRNA display (in vitro virus) to cDNA display-, Naoto Nemoto, Yuuki Mochizuki, Shingo Ueno, SEIBUTSU BUTSURI, 53, 250-253 (2013)
- High-throughput screening of biomolecules using cell-free gene expression systems, L.E. Contreras-Llano and C. Tan, Synthetic Biology, 3 (1),ysy012 (2018)
- Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity, E.T. Boder, K.S. Midelfort, and K.D. Wittrup, PNAS, 97 (29), 10701-10705 (2000)
- Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, G.P. Smith, Science, 228 (4075), 1315-1317 (1985)
- cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions, J. Yamaguchi, M. Naimuddin, M. Biyani, T. Sasaki, M. Machida, T. Kubo, T. Funatsu, Y. Husimi, N. Nemoto, NAR, 37 (16), e108 (2009)
- In vitro selection and evolution of functional proteins by using ribosome display, J. Hanes and A. Plückthun, PNAS, 94, 4937-4942 (1997)
- In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro, N. Nemoto, E. Miyamoto-Sato, Y. Husimi and H. Yanagawa, FEBS Letters, 414, 405-408 (1997)
- RNA-peptide fusions for the in vitro selection of peptides and proteins, R.W. Roberts and J.W. Szostak, PNAS, 94 (23), 12297-12302 (1997)
PCT/WO2017170776A1“High-speed in vitro screening method”
Innovative drug discovery
Binding molecule profile
|Phage display||cDNA display||cDNA display +SDNC|