Construction Of Functional Nucleic Acid Probes And Their Application In Physiological Environment

With the rapid development of society and the improvement of human living standards,the human health problems have become increasingly prominent.Among them,malignant tumor has become one of the major diseases threatening human life and health.According to the National Cancer Research Report in 2019,the number of deaths caused by malignant tumors is as high as 23.91%,and the incidence rate is increasing year by year.Therefore,accurate diagnosis and efficient treatment of major diseases such as cancer are extremely urgent.It is extremly vital to develop probes for specifically recognizing disease markers and expand candidate drug molecules for disease diagnosis and treatment system,and the preparation of probes and drug molecules is based on molecular recognition and interaction.Functional nucleic acids are oligonucleotides with molecular recognition or enzyme like catalytic activity.It has the advantages of flexible and controllable design,wide target range,rapid synthesis and easy chemical modification.It can be roughly classified into three categories:the first category refers to DNA molecular fragments with molecular recognition and enzyme like catalytic activity,called DNAzyme;the second category refers to oligonucleotide fragments with the same recognition ability as antibodies,but with a wider range of target molecules,called aptamers;the third category refers to single stranded oligonucleotide probes with stem loop structure,called molecular beacon.Since it was first proposed in the 1990s,due to its excellent molecular recognition function,functional nucleic acids have attracted much attention in the disease diagnosis and treatment system,providing a new tool for accurate diagnosis and efficient treatment of diseases at the molecular level.Traditional functional nucleic acids are used as molecular tools for disease and related diagnosis and treatment,they are often limited by the corresponding physiological environment.For example,due to the interference of metal ions,protein molecules and other substances in the physiological environment,the recognition ability and detection sensitivity of functional nucleic acids to some disease biomarkers are greatly limited,and the release efficiency of some functional nucleic acid drug molecules in cells is low.Therefore,in order to solve these problems,this thesis studies from the following aspects:(1)In T-cell-based tumor immunotherapy,the necrosis of some cancer cells in tumor tissue results in the release of high concentration of potassium ions,which seriously damages the metabolism of surrounding infiltrating T cells,reduce their activity,cause ion immunosuppression,and reduce the anti-tumor activity.In order to study the phenomenon of ion immunosuppression,it is very important to accurately detect potassium ion in the corresponding physiological environment.G-quadruplex functional nucleic acids are widely used as potassium ion recognition units,but it is difficult for traditional G-quadruplex functional nucleic acids to specifically recognize potassium ion under physiological concentration of sodium ion(?150 m M).In the chapter 2,we propose to recognize K⁺by G-quadruplex with intermolecular conformational change,instead of simultaneously recognition K⁺and Na⁺by traditional G-quadruplex with intramolecular conformational change.The oligo-3(5?-TGAGGGAGGGG-3?)was successfully selected from the half length G-quadruplex rich sequence by cutting the traditional G-quadruplex structure into two parts.In the presence of 150 m M Na⁺,the correlation coefficient of K⁺(5.0 m M)selectivity of oligo-3 was 232,which laid a foundation for the selective detection of K⁺in the presence of physiological Na⁺.(2)When G-quadruplex functional nucleic acid fluorescence directly detects potassium ion in physiological environment,there are two challenges:the degradation of nucleic acid in environment and the high background fluorescence of biological spontaneous.Therefore,in the chapter 3,we first selected the half-length G-quadruplex functional nucleic acid oligo-3,which specifically recognized potassium ion under physiological conditions as the recognition unit,and modified the 3'-terminal of nucleic acid with reverse T base modification,which successfully protected against nuclease cleavage.In addition,the G-quadruplex structure embedded two-photon dye EBMVC-B was used to output the signal,which significantly reduced the interference of background fluorescence signal in complex biological system.Finally,because the whole structure of G-quadruplex is split,the binding of oligo-3 to K⁺becomes more difficult,and the linear response range of oligo-3 to K⁺is 100?M-10m M.The potassium ion is successfully detected in the physiological environment by simulating tumor microenvironment with blood.(3)Alzheimer's disease(AD)is a kind of neurodegenerative disease.The single index detection is easy to cause false positive signals and concentration of disease markers is low in the blood.Aiming at these two challenges,in Chapter 4,we adopt the strategy of electrochemical simultaneous analysis of multiple disease markers of Alzheimer's disease based on secondary structure encoded triple chain molecular switch(TMS)nanopore.The specific principle is that the TMS loop is aptamer sequence as the recognition unit to separately identify three kinds of Alzheimer's disease markers.The middle chain at the tail of the three strand is single strand,single strand ends folded into double strand and single strand ends folded into G-quadruplex.The three kinds of DNA chains have different spatial scales and are used as signal transduction chains.Because the hairpin structure of TMS is larger in size,and the?-hemolysin nanopore channel is relatively narrow,it can not enter the nanopore channel,and the current is unobstructed;when three kinds of disease markers exist,the three chain structures will open,the signal transduction chain will be released,and pass through the nanopore.Due to the different sizes of the three signal transduction chains in space,the time of passing through the nanopore will be significantly different,resulting in the current blocking at different times and the output of three distinct electrochemical signals.In addition,the biosensor based on electrochemistry has high sensitivity,so this strategy can be used to simultaneously analyze the markers of Alzheimer's disease in blood with high sensitivity and multi parameters.(4)The abundance of prostate cancer specific antigen(PSA),a marker of prostate cancer,is extremely low in the blood.In order to detect this low abundance substance,in Chapter 5,we constructed a TMS nucleic acid signal amplification probe based on human serum albumin(HSA)as internal signal amplifier for highly sensitivity detection of PSA in blood.The specific principle is that the ring part of the triple chain molecular beacon is a aptamer sequence that can recognize PSA.A small molecule fluorescent dye EBCB can be embedded into double or triple strands of DNA molecules in the stem.The tail of central chain in the stem is marked with thiol functional groups.Interestingly,we found that EBCB can bind HSA specifically and greatly enhance the fluorescence quantum yield.When we anchor the triplex molecular beacon to the surface of gold nanoparticles through Au-S bond,the gold nanoparticles will largely quench the fluorescence of the dye EBCB.When PSA exists,it will bind the ring part of molecular beacon,simultaneously causing conformational change;this results then make the triple chain be switched on and the concurrently release the dye with restoring the fluorescence.And lastly,the free EBCB will immediately embed into the HSA in the serum and result in the amplification of cascade signal.for highly sensitive detection of PSA in serum.(5)After functional nucleic acid drug CpG DNA binds to protein TLR9 receptor in cell endosomes,it also sequencely bind to a series of downstream signal molecules such as My D88 in cytoplasm for activating the whole signaling pathway.Therefore,the free CpG DNA in cells can play a better role in drug activity.In Chapter 6,CpG DNA was loaded with ceria nanoparticles,Compared with the traditional covalent crosslinking on the surface of nanoparticles,the CpG DNA loaded with ceria nanoparticles can be replaced by H₂O₂.Then,the compounds of released DNA and protein TLR9 receptor can activate downstream signal pathway more freely,which can better stimulate the maturation of antigen presenting cells(APC)and subsequent immune response.Through the combination of immunotherapy with photodynamic therapy,tumor vaccine can be generated in situ to promote immune response and attack the residual tumor tissue after photodynamic therapy,so as to avoid the recurrence of tumor tissue.The thesis focuses on functional nucleic acid as a molecular recognition tool.Through its modification and transformation,it can be applied to the physiological environment to effectively improve the accuracy and sensitivity of disease diagnosis,and further improve the efficiency of disease treatment.The design and construction of functional nucleic acid probe strategy has certain universality and potential application prospects for the accurate analysis of biomarkers in the physiological environment.