Selection And Application Of Chemically Modified Functional Nucleic Acids

Functional nucleic acids mainly include nucleic acid aptamers and enzymes.Unlike traditional nucleic acids acting as genetic information in the linear form,functional nucleic acids fold into stable tertiary structures through intramolecular forces and exert functions of binding ligands and catalysis reaction.Nucleic acid aptamers are a class of single stranded oligonucleotides with target-recognition,and researchers have obtained various high affinity aptamers that specifically bind to targets such as small molecules,proteins,bacteria,viruses,cells and so on.Aptamers have been applied in many areas of detection of molecular,isolation of protein,regulation of immunity,and targeted drug delivery.Nucleic acid enzymes are the single stranded nucleic acids with catalytic capacity,generally composed of binding domains and catalytic core.After specifically recognizing and binding substrates,enzymes can catalyze many reactions such as breaking or forming of phosphodiester linkages between substrates.Depending on diversity of the reaction conditions and substrates,enzymes are mainly used in ion detection and gene silencing.In research,scientists find that natural nucleic acids are susceptible to degradation by nucleases,they are poor stability under physiological conditions and cannot meet the experimental demands.Additionally,natural nucleic acids have fewer functional groups and relatively limited chemical diversity.To improve the biological stability and chemical diversity of nucleic acids,researchers develop a series of chemically modified nucleic acid monomers through changing the nucleic acid phosphate,sugar ring and base.With the action of polymerases,scientists have developed many unnatural functional nucleic acids molecules that are more resistant to nuclease degradation and with higher affinity to targets.When applied in the cells and living organisms,these unnatural nucleic acids have greater biological stability and a longer effect.Therefore,they have the potential values in biotechnology,disease diagnosis and drug development.In this thesis,three aspects of work have been carried out:(1)Immunoblotting is one of most common method for specific protein detection and quantification in laboratories,and it relies on the highly selective and high affinity of antibodies.Compared with antibodies,nucleic acid aptamers have several merits,including low cost of production,refolding after denaturation,easy to be chemically modified.In this part of the work,we applied fluorescence labeled aptamers in protein detection by Western blot,which simplified the operation process,saved experimental time and reduced economic cost.We chose three common tag proteins as targets,including glutathione S-transferase(GST),polyhistidine tag(His-tag),and maltose binding protein(MBP).Through in vitro selection,we isolated aptamers G1 and G2 targeted to GST,aptamers H1and H2 bound to His-tag and aptamer M1 bound to MBP.The dissociation constants of these aptamer were at the nanomolar per liter.Further,we found that the aptamer G1 could effectively discriminate the target protein from three proteins,specifically binding to the target protein GST with a higher specificity than the anti-GST antibody.Finally,we found that G2,H2,and M1 could selectively bind to the fusion proteins,which broaden the applications of these aptamers.This work suggested that the methodology developed here could be extended to other tag proteins,and the aptamers generated through such selections would represent a type of simpler and more affordable alternative affinity reagent for protein immunoblotting analysis(2)Bladder cancer is one of the highest incidence cancers in the urinary system,and surgical resection and postoperative chemotherapy are mainly clinical treatment of bladder cancer.Common chemotherapy drugs can cause serious side effects due to lack of targeting.To solve this problem,we aimed to construct a targeted drug delivery system with high efficiency and low toxicity,providing a new strategy for bladder cancer treatment.The second part in this thesis,we performed the cell-internalization SELEX and isolated the chemical modified nucleic acid aptamers targeting bladder cancer cell T24.The chemically modified Aptamer B1 could specifically recognized and internalized various bladder cancer cells.Additionally,we explored the mechanism of Aptamer B1 internalizing into cells and possible target molecular.Finally,we constructed a drug delivery complex with Aptamer B1 as targeting group,and the results suggested that this aptamer-drug complex could specifically internalized bladder cancer cells and decreased the toxic effects of chemotherapy drugs on normal bladder epithelial cells.The targeted drug delivery system had a better effect in inhibiting growth of orthotopic bladder tumor in vivo.This work developed the first chemically modified aptamer against bladder cancer cells with high specificity and internalization capacities.The auto-assembled nanotrain drug-delivery complex obviously improved the treatment effect of chemotherapeutic drugs.This work provides a new strategy for the treatment of bladder cancer,with the application potential in the clinical.(3)RNA has a wealth of structural and functional diversity,leading scientists to propose the RNA world hypothesis,which postulates early life on earth use RNA as genetic information and catalyze metabolic reactions,but whether RNA was the first or the only possible genetic polymer was debatable.Threose nucleic acid(TNA)has been found to form stable duplex structures with complementary strands of RNA or itself,the sugar ring of TNA is simpler,thus TNA has been proposed as a potential RNA precursor.Genetic information as the early life on earth requires that TNA can fold into three-dimensional structures with catalytical activity like ribozymes.In the third part of this thesis,we reported the isolation of RNA ligase TNA enzymes by in vitro selection.We identified the TNA enzyme T8-6 after 8 rounds of selection and truncation optimization,it catalyzed two RNA substrates ligation to form a new 2'-5'phosphodiester bond.T8-6 required UA|GA at the ligation junction and tolerated at other substrate positions.The reaction kinetic constant of T8-6 was 0.011 min^-1 at p H 9.0,40 m M Mg²⁺,and the catalytic activity was dependent on concentration of Mg²⁺,reaching maximal at 40 m M Mg²⁺.Additionally,T8-6 was tolerated for divalent metal ions and also exhibit lower ligation activity at Mn²⁺,Zn²⁺and CO²⁺and Cu²⁺.Finally,we ensured that generation of a hammerhead ribozyme containing a 2'-5'phosphate linkage with TNA enzyme T8-6 had equivalent cleavage activity to the chemically synthesized completed 3'-5'linkage sequence.This work isolated an RNA ligase TNA enzyme by in vitro selection for the first time,providing experimental evidence that TNA is a potential RNA precursor,and offered an alternative molecular tool of RNA ligase for the field of nucleic acid chemistry.The in vitro selection method of unnatural nucleic acids established in this work can be generalized to the selection of other molecular and promote researchers to develop more functional nucleic acids with applications in molecular detection and disease treatment and many other areas.In summary,we isolated nucleic acid aptamers for immunoblotting by in vitro selection,which greatly simplified the experimental process and imaged of the target protein on membrane in one step.Additionally,a chemically modified aptamer was generated that specifically recognized and internalized into bladder cancer cells,the constructed aptamer-drug complex showed specifical toxicity to cancer cells and significantly inhibited bladder tumor growth in vivo.This work provides a new strategy for the clinical treatment for bladder cancer.In the end,we identified the RNA ligase TNA enzyme,which provided experimental support for TNA as the potential genetic polymer of early life.All these works offer experimental basis for the development and application of functional nucleic acids,and promote the field of nucleic acids chemistry for further research.