Construction And Applications Of Chemically Modified Aptamers Based On Solid-phase Synthesis

Aptamers have been widely used in tumor diagnosis and targeted therapy in recent years due to their advantages of highly specific target recognition and precise and controllable modification sites.However,unmodified natural aptamers still face many challenges in practical applications,especially in complex environments.Natural aptamers are easily degraded by nucleases,resulting in a short half-life in vivo and insufficient time to accumulate at the tumor site.It has been observed that aptamer-drug conjugates are not only structurally unstable,they have defects such as low drug loading and off-target effects.Chemically modified aptamers have been developed to compensate for the inherent defects of natural aptamers.Pre-SELEX modification involves using chemical methods to artificially expand the library before SELEX,and then using the target to screen out chemically modified non-natural nucleic acid aptamers from the amplified library.Post-SELEX modification uses chemical theory as a guide to modify the sugar ring,base,and phosphate backbone of the natural nucleic acid aptamers that have been screened to produce non-natural nucleic acid aptamers with stable structure and enhanced affinity.Despite extensive studies on the chemical modification of natural nucleic acid aptamers has been extensively studied,there are still some unsolved problems in this field.Pre-SELEX modification has to overcome the problem of poor compatibility of nucleic acid polymerases for modified substrates.Post-SELEX may sacrifice aptamer affinity while obtaining a more stable structure.Based on the challenges faced by natural nucleic acid aptamers in complex environments inspired the development of a series of non-natural nucleic acid aptamers with novel structures were developed and applied to tumor detection and targeted therapy research in complex environments.The details are as follows:（1）Synthesize branched multivalent aptamers and explore their properties.Traditional linear nucleic acids are unstable and easily degraded by nucleases,which greatly limit their in vivo applications.In order to expand the application of natural nucleic acids in terms of structure and function,nucleic acids with various novel topological structures have attracted extensive attention of researchers,among which branched nucleic acids as various topological structural motifs are favored.However,currently branched nucleic acids are mainly non-covalently assembled through complementary base pairing between single-stranded nucleic acids.In Chapter 2,chemically synthesized novel branched nucleic acids with covalent structures from the perspective of molecular design by simple sequence changes,we synthesized branched multivalent nucleic acid aptamers.Studies have shown that branched aptamers have enhanced serum stability and increased target affinity,and have the potential to be used in complex environments such as in vivo.（2）Construction and properties of branched aptamer-drug conjugates.ApDCs are not only simple to prepare,but also the site and quantity of conjugated drugs are precisely controllable,which provides more convenience for precise targeted drug design.In addition,the advantages of no batch-to-batch variation and relative stability to the environment facilitate the large-scale preparation and storage of ApDC.However,traditional linear ApDCs also face limitations such as structural instability and low drug loading when applied in vivo.In chapter 3,we prepared branched aptamer-drug conjugates（Sgc8-20D）by solid-phase synthesis by adding branched phosphoramidite monomers.This new type of ApDC has greatly enhanced stability and target-binding ability.Cytotoxicity studies showed that Sgc8-20D had a strong killing effect on its target cells（HCT-116 cells）.（3）Application of branched aptamer-drug conjugate（AS1411-20D）in the treatment of rat glioma.Nucleic acid solid-phase synthesis technology plays an important role in the preparation of ApDCs.The efficiency of solid-phase synthesis is affected by factors such as the type of phosphoramidite monomer and the length of the target sequence.As one of the nucleoside analogs,5-fluorouridine not only has a good tumor treatment effect but also exhibits excellent stability and high coupling efficiency in nucleic acid solid-phase synthesis.The aptamer AS1411 can target a variety of tumors and is a commonly used recognition aptamer in ApDC.The sequence of AS1411 is very short and contains multiple G-rich fragments,so it has higher stability.In Chapter 4,we selected AS1411 that with a shorter sequence and a more stable structure as the recognition unit,and 5-fluorouridine as the drug base,to prepare a new branched nucleic acid aptamer-drug conjugate（AS1411-20D）by automated solid-phase synthesis technology.Due to the excellent stability and efficient tumor cell killing effect of AS1411-20D,we further applied it to the targeted therapy of living tumors and achieved a remarkable therapeutic effect.（4）Engineering AND-Gate aptamer-signal base conjugates for targeted magnetic resonance molecular imaging of metastatic cancer.The widespread clinical application of molecular imaging is greatly limited due to the lack of targeted molecular probes.Inspired by nucleic acid chemistry,in Chapter 5,we created the targeted molecular imaging probe ApSC through the programmable integration of aptamers and activatable signaling bases using solid-phase synthesis.The probe actively targets tumor-associated membrane markers through aptamer-mediated recognition,and responds precisely to acidic p H and overproduced H₂O₂to activates the signaling bases in an AND-gate manner,resulting in amplified T1-weighted MR signals.Its targeted MR imaging was validated in primary tumors and metastases.