Periodic Reporting for period 1 - PepDNA-4D (Four-dimensional self-assembly from peptides and DNA)
Período documentado: 2019-07-29 hasta 2021-07-28
1. Peptide-oligonucleotide conjugates are currently explored for applications in the applied biomedical field, but their nanoscale assembly has not yet been investigated to a significant extent. Our work focused on introducing new multiscale diversity to DNA and peptide nanotechnology through orthogonal hierarchical and emergent assembly. In this work, for the first time, peptide chain elongation was achieved from a DNA strand using enzymes. The N-terminus of a phenylalanine ester unit was linked to the DNA-fragment through a succinic acid linker. Enzymatic polymerisation was then carried out using papain under mild aqueous conditions. L-glutamic acid diethyl ester was used as a monomer for polymerisation reaction. The polymerisation reaction yielded mixture of peptides and DNA-peptide conjugates both containing up to 8-mer peptide units. The formation of DNA-peptide conjugates was confirmed by mass spectrometry, and the resulting emergent assembly studied by AFM, DLS, and SEM.
In conclusion, we were able to achieve elongation of peptide chain from the DNA strand for the first time, and we have demonstrated that this process affected its self-assembly.
2. There are many reports of DNA-peptide conjugation, and these are potentially medically useful molecules, but there are no reports of DNA-peptide hybrids in which the peptide chain is built chemoenzymatically, which would be much more environmentally friendly. This work established an effective conjugation method to link DNA and peptide initiator. It established a new synthetic method of synthesising peptides from the DNA strand utilising enzymes under mild reaction conditions. It also gave an opportunity to learn novel self-assembled nanostructures formed during the reaction. This reaction is a sustainable approach to synthesize DNA-peptide conjugates. The reaction is free from all hazardous and toxic reagents which makes it eco-friendly synthetic approach. This work also gives an opportunity for the scientific community to further explore self-assembly behaviours of DNA-peptide conjugates by varying different parameters and to develop novel conjugates using this approach.
3. Overall objective of this project-
Introduction of chemoenzymatic polymerisation to the DNA-peptide chemistry.
In conclusion, we were able to achieve elongation of peptide chain from the DNA strand for the first time, and we have demonstrated that this process affected its self-assembly.
2. There are many reports of DNA-peptide conjugation, and these are potentially medically useful molecules, but there are no reports of DNA-peptide hybrids in which the peptide chain is built chemoenzymatically, which would be much more environmentally friendly. This work established an effective conjugation method to link DNA and peptide initiator. It established a new synthetic method of synthesising peptides from the DNA strand utilising enzymes under mild reaction conditions. It also gave an opportunity to learn novel self-assembled nanostructures formed during the reaction. This reaction is a sustainable approach to synthesize DNA-peptide conjugates. The reaction is free from all hazardous and toxic reagents which makes it eco-friendly synthetic approach. This work also gives an opportunity for the scientific community to further explore self-assembly behaviours of DNA-peptide conjugates by varying different parameters and to develop novel conjugates using this approach.
3. Overall objective of this project-
Introduction of chemoenzymatic polymerisation to the DNA-peptide chemistry.
This work provides a novel method for the synthesis of the DNA-peptide conjugates. It explored a new way of synthesising peptides from the DNA strand by utilising enzymes under mild reaction conditions. It also gives an opportunity to discover novel self-assembled structures formed during the reaction.
After some exploration of coupling chemistries, the phenyl alanine ester was reacted with succinic anhydride followed by NHS ester formation of the free acid terminal of succinic acid. The resulting NHS ester was reacted with DNA-NH2 (commercially purchased) to obtain DNA-initiator. PAGE analysis was carried out to monitor the completion of the reaction. The DNA-initiator were desalted and analysed by mass spectrum to confirm the product formation. This DNA-initiator was used as a starting material for enzymatic reactions. Initially, L-glutamic acid diethyl ester was used for peptide chain elongation on DNA-initiator. Reaction was carried out in Phosphate Buffer Solution (PBS) using enzyme (papain) for elongation of peptide chain. The product obtained from the reaction was solid which was washed with water (3 times) and air dried. The products were then treated with NaOH solution to hydrolyse the ester terminal of glutamic acid. The resulting product was a clear solution. This clear solution was desalted and analysed by the mass spectrum. The mass spectrum analysis confirms that the polymerisation was successful and yielded a mixture of peptides and DNA-peptide conjugates containing up to 8-mer peptide units.
The resulting DNA-initiator was reacted with different amino acids. Alanine, glycine, histidine, lysine phenylalanine, and arginine were used for the chemoenzymatic reactions. Tripeptides [(Valine-Proline-Glycine) and Alanine-Aminoisobutyric acid-Alanine)] were also used for the enzymatic reactions. The rates of the reaction were measured using plate reader.The reactions mixtures of enzymatic reactions were used to study emergent assembly formation by AFM, DLS, and SEM.
After some exploration of coupling chemistries, the phenyl alanine ester was reacted with succinic anhydride followed by NHS ester formation of the free acid terminal of succinic acid. The resulting NHS ester was reacted with DNA-NH2 (commercially purchased) to obtain DNA-initiator. PAGE analysis was carried out to monitor the completion of the reaction. The DNA-initiator were desalted and analysed by mass spectrum to confirm the product formation. This DNA-initiator was used as a starting material for enzymatic reactions. Initially, L-glutamic acid diethyl ester was used for peptide chain elongation on DNA-initiator. Reaction was carried out in Phosphate Buffer Solution (PBS) using enzyme (papain) for elongation of peptide chain. The product obtained from the reaction was solid which was washed with water (3 times) and air dried. The products were then treated with NaOH solution to hydrolyse the ester terminal of glutamic acid. The resulting product was a clear solution. This clear solution was desalted and analysed by the mass spectrum. The mass spectrum analysis confirms that the polymerisation was successful and yielded a mixture of peptides and DNA-peptide conjugates containing up to 8-mer peptide units.
The resulting DNA-initiator was reacted with different amino acids. Alanine, glycine, histidine, lysine phenylalanine, and arginine were used for the chemoenzymatic reactions. Tripeptides [(Valine-Proline-Glycine) and Alanine-Aminoisobutyric acid-Alanine)] were also used for the enzymatic reactions. The rates of the reaction were measured using plate reader.The reactions mixtures of enzymatic reactions were used to study emergent assembly formation by AFM, DLS, and SEM.
There was no prior way of enzymatic synthesis of peptides from DNA strands. We have established it through our research findings. We have also progressed further by looking at their self-assembly.