The Application Of Unnatural Nucleic Acid Elements In The Preparation Of Biomaterials

Nucleic acids are widely used in various fields.However,compared with amino acids with multiple side chains,nucleic acids have simple components and limited functions.And they are easily degraded by enzymes or other endogenous substances in vivo for the poor stability,which limits their applications.The introduction of unnatural nucleic acids can effectively solve this problem.Unnatural nucleic acids are obtained by modifying chemical groups on the phosphates,sugar or bases of nucleic acids.These modified groups enrich the diversity of nucleic acid structures and functions.Therefore,unnatural nucleic acids have important value in detecting reagents and materials.With the development of directed evolution technology,a series of polymerases that can recognize unnatural nucleic acids have been gradually exploited,broadening the application range of unnatural nucleic acids and providing new ideas for the enzymatic synthesis of unnatural nucleic acid materials(such as DNA hydrogels).DNA hydrogel is a biological material constructed with DNA as the basic element.It not only has the skeleton structure of hydrogels but also retains the biological function of DNA.It has brought out the perfect fusion of the structure and function of hydrogel materials,and has shown a wide range of application prospects in the fields of drug delivery,cell culture and protein synthesis.In this work,we have constructed three new types of unnatural nucleic acid hydrogels using unnatural nucleic acids as structural units.First of all,the mutant polymerase SFM4-3,SFM4-6,and SFM4-9 were tested for their amplification,transcription and reverse transcription activities on sugar-modified(2’-F or 2’-OMe)nucleic acids.Besides,recognition activity of different polymerases for base-modified nucleotides(5-propargylamino)and unnatural base pairs(d TPT3TP/d Na MTP)was also tested,which laid the foundation for the enzymatic synthesis of unnatural nucleic acids.Furthermore,three covalently cross-linked unnatural nucleic acid hydrogels were constructed by enzymatically synthesizing the nucleic acid modified with propargylamine.Firstly,based on the characteristics of phi29 DNA polymerase rolling circle amplification(RCA),long-chain nucleic acids containing propargylamine modification were synthesized by RCA,and then covalently crosslinked DNA hydrogel was prepared by coupling cross-linking agent(NHS-PEG6-NHS).Secondly,according to the highfidelity properties of Q5 DNA polymerase,the nucleic acid modified with propargylamine was synthesized by PCR,and the unnatural bottlebrush primers with controllable density were prepared.The DNA hydrogel with controllable crosslinking degree was prepared by RCA or PCR.Thirdly,based on the activity of polymerase SFM4-3 to synthesize unnatural nucleic acids,nucleic acids with both sugar modification and base modification were synthesized by PCR,which expanded the skeleton composition of unnatural bottlebrush primers and provide raw materials for the preparation of multifunctional hydrogels.Based on the above experiments,since the hydrogel prepared by PCR with bottlebrush primers can carry genetic information,this study also explored the application for hydrogels in vitro transcription of RNA and protein expression in cell-free systems.Moreover,the stability of the unnatural DNA hydrogel was verified by testing the degradation rate.The construction of unnatural DNA hydrogels has laid a foundation for the development of new biological materials and the expansion of the application of unnatural nucleic acid materials.