Studies On The Construction Of Activatable Aptamer Probes Based Onfunctionalized Carbon Nanotube And I-motif Structure And Their Applications In Tumor Imaging

Cancer is a major disease that threats human life and social development seriously,and a leading cause of deaths worldwide.The incidence of cancer is increasing year by year.Early detection and treatment of cancer is the key to improve the survival rate of cancer patients and has become one of the top priorities for biothechnologycal research.With the traditional diagnostic and treatment technique,false-positive diagnostic results and toxic side effects are always happened due to the prescence of nonspecific interaction.In recent years,the rapid progress of nanotechnology and molecular engineering technology plus their continuous intercrossing with life science provide an opportunity for developing novel tumor imaging and characterization methods.On the other hand,functional nucleic acids,such as aptamers,nucleic acid enzyms and DNA polymer,provide an ideal kind of targeted recognition molecule to meet the selectivity demand in clinical applications.Aaiming at improving the specificity and sensitivity,this dissertation presentes an exploration of the use of aptamer functionalized nanomaterial and I-motif in fluorescence tumor imaging and characteriazation.The main results were listed as follows;1.A versatile activatable fluorescence probing platform for cancer cells in vitro and in vivo based on self-sssembled aptamer/carbon nanotube ensemblesBy integrating advantages of single-walled carbon nanotube and aptamers as the signal switch and the targeted recognition molecule respectively,Sgc8c was selected as the model to construct an activatable probe for tumor imaging based on fluorophore-labeled aptamer/single-walled carbon nanotube(F-apt/SWNT)ensembles.In the absence of targets,Cy5-Sgc8c firmly assembled on the surface of SWNT and displayed a quenched fluorescence.However,with the addition of target tumor cells,the binding of aptamer with the target cell could force Cy5-labeled strand apart from SWNT with an elevated fluorescence.Through flow cytometry assay and in vivo fluorescence imaging,it was found that no matter in buffer or inside tumor-grafted mice,CCRF-CEM cells could effectively activate the fluorescence signal of this probe.2.A pH-responsive activatable aptamer probe for targeted cancer imaging based on I-motif-driven conformation alterationBased on I-motif-driven conformation alteration,we present here a pH-responsive activatable aptamer probe for targeted cancer imaging.This pH-responsive activatable aptamer probe is composed of two single-stranded DNA.One was a aptamer sequence with TAMRA label(denoted as strand A),the other(strand I),being competent to work on the formation of I-motif structure,contained four stretches of the cytosine(C)rich domain and was labeled with a BHQ2.At neutral or slightly alkaline pH,strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe,termed I-motif-based activatable aptamer probe(I-AAP).Because of proximity-induced energy transfer,the I-AAP was in a“signal off' state.The slightly acidic pH enforced the strand I to form an intramolecular I-motif and then initiated the dehybridization of I-AAP,leading to fluorescence readout in the target recognition.As a demonstration,AS1411 aptamer was used for MCF-7 cells imaging.It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment.The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues.3.Selection of I-motif sequence for highly responsive pH sensorsIn order to overcome limitations of existing I-motif structure,such as widely pH response range,low formation temperature,and low pH transition midpoint,we systematical investigation the sequence composition and the formation of I-motif.A series of C-rich DNA of different sequence compositions were systematically investigated for the formation of I-motif,the ultraviolet absorption and circular dichroism spectrum were measured to determine the effect of each DNA.After the specific sequence was identied,the reposence regularity this I-motif sequence in 37?is also studied.4.I-motif generated by screening for in vivo fluorescence imagingTo address the limitations of traditional biological antibodies,we herein constructed labeling probe for in vivo tumor imaging by adopting tumor specific aptamers evoled from cell-SELEX and I-motif generated in provious work.At the weak alkali condition(pH 7.4).the aptamer sequence and I-motif sequence can form double helix structure and showed a fluorescence quenching efficiency.After incubated with target cell in weakly acidic conditions(pH 6.5),the aptamer can bind with target cell and release the I-motif sequence.On the other hand,the I-motif sequence can also form I-motif in weakly acidic conditions and facilitate the binding of aptamer and target cell.