Development And Applications Of Nucleic Acid Amphiphiles-Based Nanostructures

Because of their programmable properties and accurate molecular recognition capabilities,nucleic acid molecules have been widely used in the fields of biomedicine,environmental science,and analytical detection etc.However,natural linear nucleic acids face many challlenges in practical applications,for instances,poor resistance against enzymatic degradation and quickly elimination from body when used in vivo.In the past twenty years,nucleic acid-based nanotechnology has made great progress to strengthen the performances of nucleic acids in complex conditions.Based on the principle of strand hybridization,nucleic acids polyhedron and origami have been developed.These nanostructures were synthesized completely from nucleic acids with accurate shape and size.Apart from nucleic acids polyhedron and origami,the combination of nucleic acid with inorganic/organic nanomaterials provides another novel strategy to generate multifunctional nucleic acids nanomaterials.Spherical nucleic acid(SNAs)is an example.SNAs are three-dimensional nanostructures,typically consisting of densely functionalized and highly oriented nucleic acids covalently attached to the surfaces of spherical nanoparticle cores.Notably,these constructs define a new class of nucleic acids that have properties that markedly differ from their linear cousin.SNAs are able to enter cells efficiently to induce gene regulation and detect biological targets in live cells,as well as enable number of other important biological applications.Nucleic acid amphiphiles is a term of hydrophobic group-conjugated nucleic acids.As we know,nucleic acids are hydrophilic for the presence of negatively charged phosphate backbone.Therefore,in aqueous solution,conventional nucleic acids are dispersived.However,the strong hydrophobic molecule induces nucleic acids amphiphiles to aggregate in buffer solution.Up to now,three types of nanostructure of nucleic acid amphiphiles have been reported,including intramolecular self-assembly into double-strand nucleic acids,intermolecular self-assembly into micellar nanoparticles and assembly with other subjects to form complex.For example,hydrophobic F bases-incorporated nucleic acids can form F bases double-strand;diacyllipid-conjugated nucleic acid can self-assembly into polymeric micelles;diacyllipid-conjugated nucleic acid can anchor on the surface of cell membrane;diacyllipid-conjugated nucleic acid can bond with serum albumin to form nucleic acid-albumin complex.These nanostructures have potential in cellular imaging of targets,engineering of nanomaterials and cell membrane,and systemic delivery of chemotherapeutic drugs.Despite of these advances,there are still some key issues that have not yet been resolved in this field.For example,does fluorinated molecular beacon has distinct physicochemical properties compared to natural molecular beacon? How to precisely regulate the structural stability of lipid-based DNA micelles? Does albumin-hitchhiking can be used for the systemic delivery of chemotherapeutic drugs? How to selectively anchor nucleic acids on a particular cell membrane? To address the above mentioned questions,this thesis was shown in the following four chapters.(1)Fluorinated molecular beacons as functional DNA nanomolecules for cellular imaging.Molecular beacons(MBs)are simple,but practical,fluorescent nanoprobes widely used to detect small molecules,nucleic acids and proteins.However,some challenges still remain when MBs are employed in complex biological environments,such as instability and non-target interference.To meet such challenges,in chapter 2,we have designed and synthesized fluorinated molecular beacons(FMBs)as functional DNA nanomolecules for cellular imaging,in which the stem sequence is simply composed of artificial nucleotides with 3,5-bis(trifluoromethyl)benzene(F)as the surrogate base of natural bases.The introduction of F base into MBs significantly increases their hydrophobicity,and the stem is formed by the assembly of self-complementary base F nucleotides through hydrophobic interactions.Fluorescence studies revealed that FMBs confer improved resist ance against enzymatic degradation over conventional MBs.To demonstrate the application of FMBs for cellular imaging,we constructed an FMB probe to detect mRNA in MCF-7 cells,and the FMB was proven to be a practical nanoprobe for cellular imaging of mRN A.(2)Engineering stability-tunable DNA micelles using photocontrollable dissociation of an intermolecular G-quadruplex.Because of their facile preparation,small size(<100 nm),programmable design,and biocompatibility,lipid-based DNA micelles show enormous potential as a tool to monitor biological events and treat human diseases.However,their structural stability in biological matrices su ffers from spatiotemporal variability,thus limiting their in vivo use.Herein,in chapter 3,we have engineered stability-tunable DNA micelle flares using photocontrollable dissociation of intermolecular G-quadruplexes,which confers DNA micelle flares with robust structural stability against disruption by serum albumin.However,once exposed to light,the G-quadruplex formation is blocked by strand hybridization,resulting in the loss of stability in the presence of serum albumin and subsequent cellular uptake.This programmable regulation to stabilize lipid-based micelles in the presence of fatty-acid-binding serum albumin should further the development of biocompatible DNA micelles for in vivo applications.(3)Floxuridine homomeric oligonucleotides “hitchhike” with albumin in situ for cancer chemotherapy.Automated attachment of chemotherapeutic drugs to oligonucleotides via phosphoramidite chemistry and DNA synthesis has emerged as a powerful technology in constructing structure-defined and payload-tunable oligonucleotide-drug conjugates.In practice,however,in vivo delivery of these oligonucleotides remains a critical challenge.Inspired by the systemic transport of hydrophobic payloads by serum albumin in nature,in chapter 4,we report the development of a lipid-conjugated floxuridine homomeric oligonucleotide(LFU20)that “hitchhikes” with endogenous serum albumin for cancer chemotherapy.Upon intravenous injection into blood,LFU20 immediately inserts into the hydrophobic cave of albumin to form an LFU20/albumin complex,which accumulates in the tumor by the enhanced permeability and retention(EPR)effect and internalizes into the lysosomes of cancer cells.After degradation,cytotoxic floxuridine monophosphate is released to inhibit cell proliferation.With the advantages of ready synthesis,tunable payloads and hitchhiking with an endogenous carrier,we demonstrate LFU20 as a promising drug in cancer chemotherapy.(4)Activable lipid-conjugated oligonucleotide selectively anchors on cell membrane with elevated alkaline phosphatase level.Because of the physical,i.e.,nonselective,insertion into the hydrophobic area of cell membrane,current lipid-conjugated oligonucleotides fail to selectively anchor on cell membranes between different cell lines.In chapter 5,we address the problem by designing novel lipid-conjugated oligonucleotide,named DNA-lipid-P,in which negatively charged phosphate group was modified at the terminus of lipid tail to decrease its hydrophobicity.DNA-lipid-P shows poor affinity in anchoring on cell membrane for their weak hydrophobicity.However,alkaline phosphatase(ALP)which expressed on cell membrane removes the phosphate groups,leading to the conversion of DNA-lipid-P to DNA-lipid which has greater hydrophobicity,followed by anchoring on ALP-elevated cell membrane in situ.