Chemical communications (Cambridge, England), Jan 10, 2015
Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of ... more Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.
Microalgae-based metabolic engineering has been proven effective for producing valuable substance... more Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering. By virtue of the excellent phylogenetic and phenotypic diversity of microalgae, microalgae-based metabolic engineering has been proven effective for producing valuable substances for food, medicine, and energy in the past decade 1-5. It involves the use of cultivation techniques and genetic engineering to optimize the metabolism of microalgae by altering their biochemical pathways and introducing new pathway components into live microalgae 1,2,6,7. Popular microalgal species for metabolic engineering are Botryococcus braunii, Chlamydomonas reinhardtii, Chlorella sp., Dunaliella tertiolecta, Euglena gracilis, Nannochloropsis sp., and Spirulina as economically viable sources of bioactive compounds such as fatty acids, carotenoids, sterols, phycobilins, pectins, toxins, and halogenated compounds 1-3,6-9. The products that have been produced by these microalgal species to date include food supplements 3 , pharmaceutical drugs 3,4,10 , biodegradable plastics 7 , and biofuels 5,7. Microalgae offer a few clear advantages in metabolic engineering over plants and other microorganisms. First, microalgae require minimal environmental resources because a majority of them can grow both autotrophically and heterotrophically 7,11. Second, they have evolved to tolerate and adapt to a broad range of environments including saline and wastewater anywhere between the equator and poles 12. Finally, they are expected to be a potential solution to global warming
Cell-penetrating peptides have important therapeutic applications in drug delivery, but the varie... more Cell-penetrating peptides have important therapeutic applications in drug delivery, but the variety of known cell-penetrating peptides is still limited. With a promise to accelerate peptide development, artificial intelligence (AI) techniques including deep generative models are currently in spotlight. Scientists, however, are often overwhelmed by an excessive number of unannotated sequences generated by AI and find it difficult to obtain insights to prioritize them for experimental validation. To avoid this pitfall, we leverage molecular dynamics (MD) simulations to obtain mechanistic information to prioritize and understand AI-generated peptides. A mechanistic score of permeability is computed from five steered MD simulations starting from different initial structures predicted by homology modelling. To compensate for variability of predicted structures, the score is computed with sample variance penalization so that a peptide with consistent behaviour is highly evaluated. Our com...
Reagents and oligonucleotides All reagents were of extra pure grade and were used as received wit... more Reagents and oligonucleotides All reagents were of extra pure grade and were used as received without further purification. All primers and template DNAs were purchased from Eurofins Genomics (Japan). Demonstration of in vitro selection using E. coli extract First, to demonstrate in vitro selection using anti-flag antibody modified magnetic beads, two template sequences [a positive sequence containing FLAG tag (FLAG, 657 bp) and a negative junk sequence not containing FLAG (JUNK, 855 bp)] were prepared (Fig. 1A). Both DNA templates contained genetic components that were required for ribosome display: T7
Antimicrobial peptides are a potential solution to the threat of multidrug-resistant bacterial pa... more Antimicrobial peptides are a potential solution to the threat of multidrug-resistant bacterial pathogens. Recently, deep generative models including generative adversarial networks (GANs) have been shown to be capable of designing new antimicrobial peptides. Intuitively, a GAN controls the probability distribution of generated sequences to cover active peptides as much as possible. This paper presents a peptide-specialized model called PepGAN that takes the balance between covering active peptides anddodging non-active peptides. As a result, PepGAN has superior statistical fidelity with respect to physicochemical descriptors including charge, hydrophobicity and weight. Top six peptides were synthesized and one of them was confirmed to be highly antimicrobial. The minimum inhibitory concentration was 3.1μg/mL, indicating that the peptide is twice as strong as ampicillin.
Chemical communications (Cambridge, England), Jul 11, 2018
Correction for 'In vitro selection of electrochemical peptide probes using bioorthogonal tRNA... more Correction for 'In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection' by Tara Bahadur K. C. et al., Chem. Commun., 2018, 54, 5201-5204.
Biochemical and biophysical research communications, Jun 2, 2018
Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step ... more Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses.
Polypeptide collupse cuused b.v thc buriai oi' h},dTophobic side clrains out of cenLact with wa[e... more Polypeptide collupse cuused b.v thc buriai oi' h},dTophobic side clrains out of cenLact with wa[er is generally obscn'od as thc initial folding dynaniics ef proLeins ;yiLh more
DSCR4 (Down syndrome critical region 4) is an orphan retrotransposon-derived de-novo originated p... more DSCR4 (Down syndrome critical region 4) is an orphan retrotransposon-derived de-novo originated protein coding gene present only in hominoids (humans and great apes). Despite being located on the medically critical genomic region and abundance of evidences indicating its functionality, the role of this gene in human cells was utterly unknown. Due to absence of any prior knowledge regarding the function of DSCR4, for the first time here we used a gene-overexpression approach to discover biological importance and cellular roles of this gene. Our analysis strongly indicates DSCR4 to be mainly involved in regulation of the interconnected biological pathways related to cell migration, coagulation and immune system. We also showed that the predicted biological functions are consistent with tissue-specific expression of DSCR4 in migratory immune system leukocyte cells and neural crest cells that shape facial morphology of human embryo. Immune system and neural crest cells are also shown to...
One of the most powerful attributes of proteins is their ability to bind to and modulate the chem... more One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy) 3 2+ , an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy) 3 2+ (K d = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy) 3 2+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.
We examined the importance of aptamer usage under the same condition as the selection process by ... more We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole. We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM. We found that the addition of the common sequence, which was employed for ri...
To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeed... more To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.
The characterization of protein folding dynamics in terms of secondary and tertiary structures is... more The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
Chemical communications (Cambridge, England), Jan 10, 2015
Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of ... more Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.
Microalgae-based metabolic engineering has been proven effective for producing valuable substance... more Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering. By virtue of the excellent phylogenetic and phenotypic diversity of microalgae, microalgae-based metabolic engineering has been proven effective for producing valuable substances for food, medicine, and energy in the past decade 1-5. It involves the use of cultivation techniques and genetic engineering to optimize the metabolism of microalgae by altering their biochemical pathways and introducing new pathway components into live microalgae 1,2,6,7. Popular microalgal species for metabolic engineering are Botryococcus braunii, Chlamydomonas reinhardtii, Chlorella sp., Dunaliella tertiolecta, Euglena gracilis, Nannochloropsis sp., and Spirulina as economically viable sources of bioactive compounds such as fatty acids, carotenoids, sterols, phycobilins, pectins, toxins, and halogenated compounds 1-3,6-9. The products that have been produced by these microalgal species to date include food supplements 3 , pharmaceutical drugs 3,4,10 , biodegradable plastics 7 , and biofuels 5,7. Microalgae offer a few clear advantages in metabolic engineering over plants and other microorganisms. First, microalgae require minimal environmental resources because a majority of them can grow both autotrophically and heterotrophically 7,11. Second, they have evolved to tolerate and adapt to a broad range of environments including saline and wastewater anywhere between the equator and poles 12. Finally, they are expected to be a potential solution to global warming
Cell-penetrating peptides have important therapeutic applications in drug delivery, but the varie... more Cell-penetrating peptides have important therapeutic applications in drug delivery, but the variety of known cell-penetrating peptides is still limited. With a promise to accelerate peptide development, artificial intelligence (AI) techniques including deep generative models are currently in spotlight. Scientists, however, are often overwhelmed by an excessive number of unannotated sequences generated by AI and find it difficult to obtain insights to prioritize them for experimental validation. To avoid this pitfall, we leverage molecular dynamics (MD) simulations to obtain mechanistic information to prioritize and understand AI-generated peptides. A mechanistic score of permeability is computed from five steered MD simulations starting from different initial structures predicted by homology modelling. To compensate for variability of predicted structures, the score is computed with sample variance penalization so that a peptide with consistent behaviour is highly evaluated. Our com...
Reagents and oligonucleotides All reagents were of extra pure grade and were used as received wit... more Reagents and oligonucleotides All reagents were of extra pure grade and were used as received without further purification. All primers and template DNAs were purchased from Eurofins Genomics (Japan). Demonstration of in vitro selection using E. coli extract First, to demonstrate in vitro selection using anti-flag antibody modified magnetic beads, two template sequences [a positive sequence containing FLAG tag (FLAG, 657 bp) and a negative junk sequence not containing FLAG (JUNK, 855 bp)] were prepared (Fig. 1A). Both DNA templates contained genetic components that were required for ribosome display: T7
Antimicrobial peptides are a potential solution to the threat of multidrug-resistant bacterial pa... more Antimicrobial peptides are a potential solution to the threat of multidrug-resistant bacterial pathogens. Recently, deep generative models including generative adversarial networks (GANs) have been shown to be capable of designing new antimicrobial peptides. Intuitively, a GAN controls the probability distribution of generated sequences to cover active peptides as much as possible. This paper presents a peptide-specialized model called PepGAN that takes the balance between covering active peptides anddodging non-active peptides. As a result, PepGAN has superior statistical fidelity with respect to physicochemical descriptors including charge, hydrophobicity and weight. Top six peptides were synthesized and one of them was confirmed to be highly antimicrobial. The minimum inhibitory concentration was 3.1μg/mL, indicating that the peptide is twice as strong as ampicillin.
Chemical communications (Cambridge, England), Jul 11, 2018
Correction for 'In vitro selection of electrochemical peptide probes using bioorthogonal tRNA... more Correction for 'In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection' by Tara Bahadur K. C. et al., Chem. Commun., 2018, 54, 5201-5204.
Biochemical and biophysical research communications, Jun 2, 2018
Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step ... more Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses.
Polypeptide collupse cuused b.v thc buriai oi' h},dTophobic side clrains out of cenLact with wa[e... more Polypeptide collupse cuused b.v thc buriai oi' h},dTophobic side clrains out of cenLact with wa[er is generally obscn'od as thc initial folding dynaniics ef proLeins ;yiLh more
DSCR4 (Down syndrome critical region 4) is an orphan retrotransposon-derived de-novo originated p... more DSCR4 (Down syndrome critical region 4) is an orphan retrotransposon-derived de-novo originated protein coding gene present only in hominoids (humans and great apes). Despite being located on the medically critical genomic region and abundance of evidences indicating its functionality, the role of this gene in human cells was utterly unknown. Due to absence of any prior knowledge regarding the function of DSCR4, for the first time here we used a gene-overexpression approach to discover biological importance and cellular roles of this gene. Our analysis strongly indicates DSCR4 to be mainly involved in regulation of the interconnected biological pathways related to cell migration, coagulation and immune system. We also showed that the predicted biological functions are consistent with tissue-specific expression of DSCR4 in migratory immune system leukocyte cells and neural crest cells that shape facial morphology of human embryo. Immune system and neural crest cells are also shown to...
One of the most powerful attributes of proteins is their ability to bind to and modulate the chem... more One of the most powerful attributes of proteins is their ability to bind to and modulate the chemistry of cofactors and prosthetic groups. Here, we demonstrated the ability of an artificial nucleic acid (an aptamer) to similarly control the functionality of a non-biological element. Specifically, we selected an RNA aptamer that binds tris(bipyridine) ruthenium (II), Ru(bpy) 3 2+ , an inorganic complex that has attracted intense interest due to its photoredox chemistry, including its ability to split water by visible light. We found that a newly discovered aptamer strongly and enantioselectively binds Λ-Ru(bpy) 3 2+ (K d = 65 nM) and, in doing so, selectively suppresses deactivation via energy transfer, thereby elongating the lifetime of its photo-excited state by four-fold. The ability of the aptamer to enhance this important aspect of Ru(bpy) 3 2+ chemistry illustrates a broader point concerning the potential power of combining in vitro-created biomolecules with non-biological reactants to perform enhanced chemical reactions.
We examined the importance of aptamer usage under the same condition as the selection process by ... more We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole. We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM. We found that the addition of the common sequence, which was employed for ri...
To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeed... more To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.
The characterization of protein folding dynamics in terms of secondary and tertiary structures is... more The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
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Papers by Takanori Uzawa