Research On Conjugated Polymer-based RNA In Vitro Transcription Detection And Alginic Acid Hydrogel Peritumoral Glucose Imaging Method

RNA and glucose are two important basic substances in life activities,which play pivotal roles in many life activities.Firstly,this paper established a highly sensitive method for real-time detection of RNA synthesis in vitro transcription based on cationic conjugated polymer-mediated fluorescence resonance energy transfer;Secondly,a method for imaging peritumoral glucose in mice with alginate hydrogel containing peroxyoxalate chemiluminescence nanoparticles was designed.1.Monitoring of RNA in Vitro Transcription Based on Cationic Conjugated Polymer-mediated Fluorescence Resonance Energy Transfer MethodRNA is the cornerstone of central dogma,and it carries genetic information and involves in regulation of gene expression,which plays an extremely indispensable role in cellular pathways and disease diagnosis.Monitoring and controlling RNA transcription will be of great helpful for studying the mechanisms of modulation and inhibitor of RNA polymerase.In this study,based on the light-harvesting ability and optical amplification of cationic conjugated polymer poly(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-1,4-phenylene)bromide(PFP),real-time detection of RNA in vitro transcription was achieved by its fluorescence resonance energy transfer with fluorescent RNA aptamer complex.Firstly,this paper designed DNA template,which consisted of four sequences:T7 promoter,RNA of interest(ROI),a highly active hammerhead ribozyme(HHR),and RNA aptamer successively.RNA transcription proceeded in the presence of T7 RNA polymerase,and the active of HHR resulted in the post-transcriptional cleavage of the synthesized RNA strand,ultimately leading to the formation of two products:the ROI and the HHR+RNA aptamer.Then the DFHBI bound to the RNA aptamer sequence forming complexes,and thereby induced a fluorescence signal.When the cationic conjugated polymer PFP was added to the reaction system,PFP was used as an energydonor,RNA aptamer/DFHBI as an energy acceptor,and FRET occurred,which could enhance the fluorescence of RNA aptamer/DFHB.Experimental results showed that the fluorescence intensity of PFP-Spinach2/DFHBI was more than 3 times higher than that of Spinach2/DFHBI complex,in the presence of PFP.This system was used for quantitative analysis of transcription products in vitro,and the detection limit was 0.29 nM,which was more than 9 times lower than the detection limit(2.8 nM)of alone Spinach2/DFHBI by fluorescence method.In addition,the system performed real-time detection of RNA transcription in vitro,and the effect of inhibitors on the transcription process was studied by adding transcriptional inhibitors.We observed the FRET from PFP to RNA aptamer/DFHBI for the first time,which extended the scope of application of PFP.In addition,this method showed great potential in modulation and inhibition of RNA synthesis,providing a research means for the influence of inhibitors on RNA synthesis and clinical research on drugs related to polymerase.2.Imaging Study of Peritumoral Glucose Based on Chemiluminescent Nanoparticles Doped Alginate HydrogelGlucose is the main energy source for cell metabolism,providing energy for cell growth.Studies have shown that the energy metabolism of normal cells is significantly different from that of tumor cells,which rely on anaerobic glycolysis to provide energy.Moreover,compared with normal cells,tumor cells need to take up and consume more glucose to maintain energy demand due to rapid growth.Therefore,the level of glucose in vivo can reflect the relevant status of tumor cells,and the detection of glucose levels in vivo can provide particularly important information for the diagnosis and treatment of tumors.This paper constructed a method for imaging glucose around the tumor with alginate hydrogel containing chemiluminescent nanoparticles.Firstly,Pluronic F-127 encapsulated bis(2-carbopentyloxy-3,5,6-trichlorophenyl)oxalate(CPPO)and rubrene to form peroxyoxalate chemiluminescence nanoparticles(POCL NPs).Then,glucose oxidase(GOx)was dissolved in the aqueous solution of nanoparticles and mixed with an aqueous solution of alginate to form a POCL-GOx-Alginate solution.When Ca2+was added,the hydrogel was formed,namely POCL-GOx-Alginate/Ca hydrogel.When glucose permeated into the reaction system,it would be oxidized by GOx and hydrogen peroxide(H2O2)was produced.Peroxyoxalate chemiluminescence(POCL)shows high selectivity toward H2O2.CPPO,H2O2 and rubrene forms POCL reaction system and can undergo chemiluminescence.Experimental results showed that this POCL-GOx-alginate/Ca hydrogel responded well to glucose in vitro,which could quantitatively determine the concentration of glucose in vitro.And when chemiluminescence hydrogel was injected into tumor-bearing mice,it applied for image peritumoral glucose.The detection system with alginate/Ca hydrogel skeleton for in vivo imaging analysis prevented the diffusion of chemiluminescent reagents,which could help to improve the chemiluminescence signal.Sodium alginate has good biocompatibility and biodegradability and has been used as a carrier for subcutaneous administration,so this experimental design has the potential for clinical application.Furthermore,this chemiluminescence nanoparticle-oxydase/enzymes-alginate/Ca hydrogel system provides a platform for in situ imaging of substrates of other oxydase enzymes in vivo.