Synthesis And Applications Of Novel Lipid-Conjugated Nucleic Acids

With the in-depth study of the chemistry and biology of nucleic acids,it has been found that nucleic acids were not only important carriers for storing and transmitting genetic information,but also possess many biological functions such as molecular recognition and catalytic activity.The nucleic acids with special function were termed as "functional nucleic acids",including nucleic acid aptamers and deoxyribozymes.In the past two decades,functional nucleic acids have been widely used in biosensing,bioimaging and biomedical research due to their advantages,such as easy synthesis,good selectivity and strong binding ability.However,because of the low biostability and relatively single function,the application of functional nucleic acids in complex biological systems faces many challenges.To overcome these problems,a variety of functional nucleic acid analogs have been developed.And the lipid-conjugated nucleic acid is an important category of them.Lipid-conjugated oligonucleotide is a kind of nucleic acids amphiphiles that composed of two segments: the lipid tails as hydrophobic group and the negatively charged oligonucleotides as hydrophilic moiety.Because of the strong hydrophobicity of lipid tails,lipid-conjugated oligonucleotides can self-assemble into aggregated nanostructures in buffer solution,for instances,DNA micelles.DNA micelles show enormous promising in biochemical sensing,drug delivery and biomedicine applications due to their advantages,such as well-defined structure,tunable size,the programmable design of DNA and biocompatible.Apart from the intermolecular self-assembly into DNA micelles,lipid-conjugated nucleic acids can also insert into the cell membranes by the hydrophobic interaction between lipid tails and the hydrophobic area of cell membranes.As such,nucleic acids were anchored on the cell surface.Membrane-anchored nucleic acids can be used to monitor the changes of metal ions in cellular microenvironments and the dynamic and transient molecular encounters on the live cell membrane.Despite of this advancement,there are several challenges that limit the further applications of lipid-conjugated nucleic acids.For instances,how to precisely control the self-assembly of lipid-conjugated nucleic acids? Is there any possibility to construct DNA micelles with functional hydrophobic core? How to precisely control the cell membrane anchorage of lipid-conjugated nucleic acids? How to develop a targeted drug delivery platform by using of membrane-anchored aptamer? To overcome these challenges,this doctoral thesis was displayed as follows:(1)Creating new functional building blocks that expand the versatility of nanostructures depends on bottom-up self-assembly of amphiphilic biomolecules.Inspired by the unique physicochemical properties of hydrophobic perfluorocarbons,coupled with the powerful functions of nucleic acids.In chapter 2,we reported the synthesis of a series of diperfluorodecyl-DNA conjugates(PF-DNA)which can efficiently self-assemble into micelles in aqueous solution.On the basis of the micelle structure,both target binding affinity and enzymatic resistance of the DNA probe can be enhanced.In addition,based on the hydrophobic effect,the PF-DNA micelles(PFDM)can actively anchor onto the cell membrane,o ffering a promising tool for cell-surface engineering.Finally,the PFDM can enter cells,which is signi ficant for designing carriers for intracellular delivery.The combined advantages of the DNA micelle structure and the unique physicochemical properties of per fluorocarbons make these PFDM promising for applications in bioimaging and biomedicine.(2)In chapter 3,enzymatically triggered self-assembly of DNA amphiphile has been developed by dephosphorylation-induced increase of hydrophobicity.Phosphorylated lipid-conjugated nucleic acid(DNA-lipid-P)has four phosphate groups at the terminus of lipid tails to decrease their hydrophobicity.Therefore,DNA-lipid-P cannot self-assemble into aggregated nanostructures to some extent.However,alkaline phosphatase(ALP)converts DNA-lipid-P to lipid-conjugated oligonucleotides(DNA-lipid)by enzymatic dephosphorylation.The generated DNA-lipid has greated hydrophobicity than DNA-lipid-P thus can self-assemble into aggregated DNA-based nanostructures.Since ALP is a critical index in some cancer cells,ALP-responsive self-assembly of DNA amphiphiles show promising in drug delivery,molecular imaging and disease diagnose.(3)In chapter 4,a novel lipid-conjugated nucleic acid(DNA-lipid-PC-T4)has been developed by introducing photocleavable nucleic acid chain into the hydrophobic tails of lipid-conjugated nucleic acid through PC connector.DNA-lipid-PC-T4 shows poor cell membrane anchorage for their weak hydrophobicity.However,after sequential photo-and ALP-responsive cleavages,DNA-lipid-PC-T4 was converted to lipid-conjugated nucleic acid(DNA-lipid)which has stronger hydrophobicity than DNA-lipid-PC-T4;the generated DNA-lipid then anchors on the cell membranes of interest in situ.Because of the stepwise dual facto rs-based control,DNA-lipid-PC-T4 performs more precise anchorage on the cell membranes compared to DNA-lipid-P.(4)Exosomes(Exos)are nanoscale natural vehicles for transporting biomolecules to facilitate cell-to-cell communication,indicating a high potential of them for delivering therapeutics.To improve their delivery capacity,a simple,noninvasive,and efficient strategy for functionalizing Exos with effective targeting ligands as well as elucidation of the cellular uptake mechanism of these functi onalized Exos was found be to necessary,but remained a challenge.In chapter 5,we used diacyllipid-aptamer conjugates as the targeting ligand to develop an aptamer-functionalized Exos(Apt-Exos)nanoplatform for cell type-specific delivery of molecular therapeutics.The cellular uptake mechanism of Apt-Exos was investigated in details,and distinct behavior was observed in comparison to free Exos.By combining the excellent molecular recognition capability of aptamers and the superiority of Exos as natura l vehicles,Apt-Exos can efficiently deliver molecular drugs to target cancer cells,providing a promising delivery platform for cancer theranostics.