In Vitro Selection Of Xeno-Nucleic Acid(XNA) Aptamers And Nanopore Sequencing Of XNA

Nucleic acids act as genetic materials for information storage and transmission,and are molecular basis of all living organisms.Natural nucleic acids can not only form duplex structures through Watson-Crick base pairing,but are also capable of folding into more complicated three-dimensional structures with binding abilities and catalytic activities.These functional nucleic acids in biology play key roles in gene expression regulation and biochemical reaction acceleration,and could also be generated in the laboratory and developed into diagnostic and therapeutic tools.However,the development of functional nucleic acids for biomedical applications was often hindered by the fact that natural DNAs and RNAs are easily degraded by ubiquitous nucleases in vivo.Chemically modified nucleic acids,or xeno-nucleic acids(XNAs)not only show superior resistance to nuclease degradation,but also increase the chemical diversity of nucleic acids.Therefore,it is of high demand to develop diverse functional XNA molecular tools for the fields of chemical biology and synthetic biology.The main work of this thesis focuses on the in vitro selection of XNA aptamers and nanopore sequencing of XNA polymers.The isolation of functional XNAs through in vitro evolution and nanopore sequencing of XNAs both require XNA polymerases that can recognize unnatural nucleic acid substrates.We tested several engineered DNA polymerases for their activities in synthesizing and reverse transcribing long XNA polymers,including three TNA(threose nucleic acid)polymerases and two FANA(2'-deoxy-2'-fluoroarabinonucleic acid)reverse transcriptases.The results showed that Kod-RI is the most efficent polymerase for TNA polymer synthesis,and RT521 K is the most efficient in FANA reverse transcription.Blockade of immune checkpoint protein interactions has proven effective in clinical cancer treatment.At present,the clinical treatment of inhibiting the interaction between programmed cell death protein 1(PD-1)and its ligand PD-L1 is mainly relied on monoclonal antibodies,which are often associated with high production costs and frequent immune-related adverse effects.Herein TNA aptamers were selected in vitro to bind PD-L1 protein and to inhibit its interaction with PD-1.The blockade efficiency of the optimized TNA aptamer was over 50% and could effectively inhibit PD-L1 binding to the cell overexpressing PD-1 on its surface.Besides,tumor-bearing mice experiments indicated that TNA aptamer can successfully target CT26 tumor cells in vivo and suppress tumor growth via immune activation.Such PD-L1 antagonistic TNA aptamers may represent an attractive XNA-based alternative inhibitor for cancer immunotherapy.FANA can form stable hybrid double-stranded structure with RNA and has been intensively studied in molecular medicine and synthetic biology.Small interfering RNA with FANA modications has shown superior gene silencing efficiency.Additionally,FANA could also fold to form tertiary structures with binding affinities and catalytic activities,although it contains a distinct sugar moiety compared with natural DNA and RNA.Conventional DNA sequencing methods cannot be directly applied to FANA sequencing,because the polymerases used in sequencing reactions do not recognize FANA strands.Nanopore sequencing is a label-free sequencing technique that does not require amplification and could achieve single molecule resolution.In this work,we collaborated and demonstrated the direct sequencing of FANA using Nanopore-Induced Phase Shift Sequencing(NIPSS).Firstly,three FANA homopolymers were synthesized and showed well-distinguished pore blockage signals in a Mycobacterium smegmatis porin A(Msp A)nanopore.Then several chimeric DNA-FANA strands were constructed,which contained a DNA drive-strand and a FANA strand to be sequenced.Direct FANA sequencing was achieved using phi29 DNA polymerase as a driving enzyme on Msp A nanopore.The statistical results indicated that Msp A nanopore could clearly distinguish between FANA and DNA strands with the same sequences.These results represent the first example of direct sequencing of a sugar-modified XNA and suggest that the developed NIPSS platform could be applicable to the direct sequencing of a wide variety of XNAs as well as other biopolymers.In summary,XNA aptamers towards immune checkpoint proteins were first generated by in vitro selection,and such XNA aptamers were demonstrated to be effective in blocking PD-1/PD-L1 interaction in vitro and inhibiting tumor growth in vivo.Additionally,direct sequencing of XNA polymers were achieved using nanopore sequencing technology.FANA strands exhibited distinct signals in Msp A nanopore compared to DNA strands with the identical sequences.Collectively,this work provides important foundations and implications for the further development of XNA-based molecular tools and medicines.