Synthesis Of PNA Monomers/chains And Construction Of PNA-based Nanostructures

Nanobiotechnology is an interdisciplinary field combining biological principles with physical and chemical procedures to generate nanostructures with specific functions and new properties,it has broad application in medicine and health,it has shown great prospective future in related industry.Many developed countries have made it a priority in their centennial scientific research plan and related projects are supported with more weight as compared to others.DNA/RNA and protein/peptide are two most widely used and the most important building blocks in the field of nanobiotechnology.Due to their physicochemical properties,these nanostructures may have some drawbacks.For example,DNA/RNA nanostructures are not stable in the absence of cation ions;structures from both DNA/RNA and protein/peptide can be degraded by some enzymes within cells.PNA is an artificially synthetic DNA analogue.The nanostructures from PNA gain the advantages of both DNA/RNA-and protein/peptide-based nanostructures,more importantly,unfavorable properties from either disappear in PNA-based nanostructures.With a promising future,PNA nanotechnology is gaining more and more attention;however,the field is still at its very early stage of development.Large scale and high throughput synthesis of PNA is crucial for PNA-based nanotechnology.At present,it remains a bottleneck for the development of the field,the development of more efficient PNA synthesis techniques is urgently needed.In this thesis,we have explored a method for efficient synthesis of PNA monomers.Then we synthesized several PNA chains from our synthesized PNA monomers.The PNA chains were designed to produce some representative PNA nanostructures.These structures are expected to have important potential applications.The main content of the dissertaion are as follows:1)A brief introduction on PNA is presented.The history of PNA monomer design and synthesis is described first;then the rules on the design of PNA chains and their synthesis are introduced;and then a brief review on the current research status on PNA-based nanostructures and the most used characterization techniques is followed;finally,the significance of this study and a summary of the thesis is presented.2)An efficient route for synthesizing four Fmoc/Boc-protection PNA monomers is introduced.From the synthesized PNA monomers,we designed and synthesized a PNA chain which can fold into a PNA G-quadruplex structure.Through click chemistry on two short chains that also serve as controls,we showed that the designed PNA chain can form PNA G-quadruplex structure.Click chemistry can be used to further turn the designed linear PNA chain into a circle;as a result,the circular PNA G-quadruplex structure is more stable than the one from a linear chain.3)Inspired by the structure of Holliday junction,we designed and synthesized PNA 4-way and 3-way junctions.The PNA junctions were characterized by fast protein liquid chromatography(FPLC),MALDI-TOF mass spectrometry and atomic force microscope(AFM),we concluded that the desired nanostructures with defined shape and stoichiometry as expected are successfully constructed.Notably,these PNA junctions are stable in pure water without adding cations as revealed by corresponding UV melting curves.4)We further designed and synthesized PNA-Peptide 3-way junctions,in the middle of which has cavity.We synthesized nine PNA chains(PNA 1-9),for each chain,N-terminus was capped with 4-pentynoic acid and C-terminus was modified by lysine-N3.One,three or five alanine residues were inserted into each of PNA chains.The nine PNA chains can self-assemble into three PNA-Peptide 3-way junctions with variable sizes of cavity.Through click chemistry,MALDI-TOF mass spectrometry and AFM,we demonstrated that the PNA junctions with variable sizes of cavity were successfully constructed.Further control experiments from designed random control chains confirmed our conclusions.Finally,all PNA 3-way junctions with different cavities are stable in pure water without adding cations as revealed by corresponding UV melting curves.