Molecular Engineering And Application Research Of Unnatural Nucleic Polymerases

Central dogma is the delivery rules of genetic information in nature,which mainly expounds the transfer law of genetic information between nucleic acids and protein.Central dogma is one of the most important laws of modern biology,which plays a great role in exploring the essence and universal law of life phenomena,and greatly promotes the development of modern biology.In the central dogma,nucleic acids（DNA and RNA）are the carriers of genetic information and polymerases are the tools of genetic information transmission.In recent years,with the development of biology,the central dogma also needs to be supplemented and improved,and one of the main directions of expansion is to develop unnatural nucleic acids.Unnatural nucleic acid is a class of nucleic acid molecules with unnatural backbone or nucleic acid bases.However,the application of unnatural nucleic acids and the engineering of unnatural nucleic acid polymerases still face many challenges,such as the design of new unnatural nucleic acid,the engineering of unnatural nucleic acid polymerase with different activities,the application of unnatural nucleic acid and unnatural polymerase in vitro and in vivo.This research mainly focuses on the molecular engineering and application of unnatural nucleic acid polymerases.Exploring the activity of template-independent terminal deoxynucleotide transferase（TdT）on unnatural base nucleotides d TPT3TP and d Na MTP,and developing related applications based on this activity;The engineering of TdT was carried out to improve its activity of modified nucleotides with a 3’reversible protective group,and a preliminary attempt of de novo DNA synthesis was also made.Tth was engineered to improve its activity of sugar 2’modified triphosphates and ribose triphosphates.Based on the wild type of TdT,the research explored the efficient template-independent incorporation of nucleotides of d TPT3 and d Na M.In view of the activity of the preliminary test,factors such as metal ions,reaction buffer and whether pre-incubation was optimized in this study.Under optimized conditions,it was shown that TdT embedded d TPT3TP and d Na MTP into DNA oligonucleotide chains.Further study found that under optimized conditions,TdT also has very good integration activity for various d TPT3 derivatives d TPT3^Amand d TPT3^Biotin.On this basis,a series of orthogonal labeling techniques for various functional DNA were developed.The high efficiency fluorescence and biotin labeling was realized without affecting the original function of these DNA.Finally,based on the high recognition activity of TdT for d Na MTP and d TPT3TP,a strategy for enzymatic preparation of DNA containing UBP at any site within the sequence was developed.This strategy makes it possible for the laboratory to quickly obtain arbitrary DNA containing UBPs,which is of great value for the development and application of unnatural base pairs and artificial life.We also demonstrate the efficient TdT incorporation of d TPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof.The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.Although TdT is capable of incorporating the unnatural base triphosphates,it can hardly utilize the 3’reversibe protected triphosphate substrate.In order to achieve enzymatic DNA de novo synthesis,it is necessary to use TdT to incorporated 3’reversibe protected triphosphates in a template-independent manner,and to accurately synthesize specific sequences of oligonucleotides through the deprotection-incorporation cycles.In this study,a highly catalytic3’-NH₂-d NTPs mutant C-11 is obtained by rational engineering（site-specific mutation,fusion protein,truncated mutant）and semi-rational engineering（site-specific saturation mutation）.At 37℃for 2 min,the incorporating efficiency of 3’-NH₂-d NTPs reachs almost 100%.Finally,we try to use mutant C-11 to explore DNA de novo synthesis by enzymatic method.TdT is a template-independent polymerase,while Tth is a template-dependent polymerase.In this study,we continue to modify Tth to achieve template-dependent synthesis of unnatural nucleic acids.The Taq mutant SFM4-3/4-6/4-9,which can efficiently synthesize sugar 2’-F and2’-OMe modified triphosphates,was obtained through phage display in the laboratory in the previous stage.Considering the high homology of Tth and Taq,this study transplantes the homology of amino acid mutation site of SFM4-3/4-6/4-9 into Tth.A series of mutant strains were obtained,in which ss Tth M1-10 could not only efficiently transcribe into RNA and 2’-F-RNA using DNA as a template,but also efficiently reverse transcribe into DNA using RNA and2’-F-RNA as a template,and at the same time significantly improve the transcription and reverse transcriptional activities of 2’-OMe substrates.The study also systematically characterized ss Tth M1-10.The development of unnatural nucleic acid polymerase provides a new idea and method for the application of unnatural nucleic acid.