Amine-functionalized Fluorescent Carbon Dots For RNA Detection And Imaging

Ribonucleic acids?RNA?are responsible for a wide range of functions in biological systems,from the central medium of genetic message?messenger RNA?or a vector for genetic information of RNA viral to the regulation of transcription and translation?microRNAs?,the construction of cellular structures?ribosomal RNA?,transportation?transfer RNA?and catalysis?ribozyme?,thus RNA is the most versatile biomacromolecules.These functions are closely linked to the spatial-temporal expression and distribution in the cells,therefore,quantitative detection and long-term real-time imaging of RNA are of great significance.Fluorescent probes are important tools for RNA research,because they not only enable quantitative detection,but also can real-time monitor the dynamic changes of RNA in the cells.Currently,the reported RNA fluorescent probes include small organic molecules,fluorescent metal nanoclusters and semiconductor quantum dots.However,these probes have the drawbacks of complicated and time-consuming synthesis steps,poor photostability and high biotoxicity,which cannot meet the needs of long-term real-time imaging.The above shortcomings could be overcame by fluorescent carbon dots?CDs?.As a promising carbon-based nanomaterial,CDs possess the advantages of easy preparation,small size,good water solubility,adjustable emission wavelength etc.,and thus are considered as non-toxic alternatives for organic fluorescent molecules and quantum dots.Consequently,CDs have great application potentials in the fields of analytical detection and real-time imaging of biomolecules.Presently,there are mainly two ways for the applications in the above fields.One is acting as an energy donor to construct a fluorescence resonance energy transfer?FRET?system with other fluorophores or quenchers,the another one is surface modification with target molecules,both of which require surface modification of CDs.Existing methods for surface modification include non-covalent and covalent modifications.Among them,non-covalent modification,mainly including electrostatic bonding,?-?stacking,hydrogen bonding and coordination,is simple in operation but is limited by its poor selectivity.The covalent modification not only increases the operation steps,but also exhibites low modification efficiency and possible change of the fluorescence properties of CDs,which greatly limits the practical application.Given the above problems,herein,novel CDs-based RNA fluorescent probes were constructed via modification-free strategy.First,with the help of liposomes,a strategy involving the modification-free construction of FRET system based CDs was developed and applied to the highly sensitive and selective detection of microRNAs?miRNAs?.In addition,an in situ synthesis method for preparation of CDs with targeting ability was established.To verify the feasibility of the proposed method,the CDs with ability to long-term targeting image RNA were prepared only via screening raw materials.The main points of our work in this thesis are summarized as follows:1.Preparation of FRET soft nanoballs for miRNA detection.The difficulties on the surface modification resulted in the insufficient application of CDs in the FRET.With the help of liposomes,the CDs?FRET donor?and BHQ-2?acceptor?at high concentrations were simultaneously entrapped into the same interior of liposomes to construct a localized FRET system,namely FRET soft nanoballs?fSNBs?.Combination with the duplex specific nuclease-assisted target cycling amplification strategy,phospholipase A₂?PLA₂?and fSNBs,a dual signal amplification method was established and applied to highly sensitive and selective detection of microRNA-141?miR-141?.As the concentration of miR141 increased,the fluorescence intensity of the CDs enhanced linearly in the range of 0.025-10 nM,with a detection limit of 16.5 pM.Compared with the detection method without FRET,namely CDs-SNBs,the detection limit of our method is reduced by 515 times.In addition,our method possess versatility,high selectivity and good accuracy,thus it not only can highly recognize miRNA sequences with single base differences,but also can be applied to the detection of miRNAs in cell samples.Compared with the CDs-based FRET system previously reported,our method is not limited by the surface functional groups of CDs.Furthermore,it can avoid the disadvantages of complicated operation,low efficiency,and changes in the fluorescence performance of CDs caused by covalent modification.2.In situ preparation of label-free CDs with RNA targeting ability.An in situ preparation method of label-free CDs with targeting ability was proposed through purposely screening raw materials according to the structural features of the corresponding targeting probes.First,by comparing the molecular structures of all RNA-specific dyes,the common structural features were summarized,namely benzene ring,nitrogen-containing heterocycle,and amines.According to this common features,phenyldiamine and triethylenetetramine?TETA?,which possess benzene rings and amines,and tend to form nitrogen-containing heterocycle,were selected as precursors to prepare CDs by simple hydrothermal method.Only the CDs?m-CDs?prepared by m-phenylenediamine and TETA exhibited strong photostability,low biotoxicity,and excellent RNA selectivity in cells and solutions.The results from the infrared spectroscopy,X-ray photoelectron spectroscopy,nuclear magnetic resonance spectroscopy and high resolution mass spectrometry indicated that isoquinoline and amines hang on the surface of m-CDs.Interestingly,these structures are also present in some RNA-specific dyes.Based on the results of nuclear magnetic resonance spectroscopy and zeta potential,we speculated that m-CDs bind to RNA mainly through isoquinoline structure and electrostatic interaction.In addition,the surface modification of non-RNA specific CDs with small organic molecules containing isoquinoline and amines endow the CDs with RNA targeting ability,indicating that the isoquinoline and amine structures are significant to RNA targeting.Finally,according to the literatures,the formation mechanism of isoquinoline structure was deduced.3.CDs-based real-time monitoring of dynamic changes of RNA in living cells and its biological effects.Due to the poor photostability and high biotoxicity,the existing RNA-specific fluorescent probes fail to long-term image cellular RNA and provide information of RNA dynamics in cellular physiological processes.However,this information is critical to reveal the functions of RNA and the close relationships between their dynamic changes and some important cellular processes.It demonstrated that m-CDs possess excellent photostability,biocompatibility,RNA specificity and versatility.The m-CDs meet the demands not only on the long-term imaging of cellular RNA,but also on RNA imaging in the cultured cells,plant tissues and animal tissues.The m-CDs were successfully used to long-term real-time monitor?up to 3 days of monitoring?RNA dynamic changes during three different cellular physiological processes,including cell mitosis,drug-induced cell death,and cell proliferation.The studies on the biological effects of m-CDs in vitroand in vivo can provide a basis for drug discovery,target search and possible biological hazards,therefore,we also explored the internalization pathway of m-CDs in cells,and toxicity,distribution and metabolic pathways in the zebrafish embryos.The m-CDs entered into the cells mainly through the caveolin-mediated endocytic pathway,and then reached the perinuclear region through the microtubule-mediated transport pathway,finally reached the nucleoli through the nuclear pore complex.In addition,m-CDs had no significant effects on the development,incubation rate and survival rate of zebrafish embryos within a certain concentration range.After entering into the body of the zebrafish larvae,m-CDs were mainly distributed in pigment cells of skin,eyeballs,pronephros,intestine,liver and blood,and were clear out from the body after 48 hours,indicating the good biosafety of m-CDs.According to the biodistribution,we speculated that the metabolic pathway of m-CDs mainly involved in two routes,one is mouth?intestine?cloaca;the other is skin/intestine?blood circulation?pronephros/liver?cloaca.In summary,we focused on the detection and imaging of RNA,and proposed a modification-free strategy to construct a CDs-based fluorescent probe for extracellularly sensitive and selective detection of miRNA,and intracellularly long-term imaging of total RNA.Additionally,the biological effects of m-CDs were investigated from cellular to individual levels.This study provides a safe and powerful tool for RNA research,and also offers new insights for the further application of CDs in targeting imaging and biochemical analysis.