· An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination

Person in charge: Wang Yueyao

 Threose nucleic acid (TNA), a potentially natural derivative of RNA, has garnered considerable interest because of its chemical simplicity, base pairing property and capability to form ligand-binding structures. Here we report the in vitro selection of RNA-cleaving TNA enzymes and demonstrate the selective gene silencing application of one such TNA enzyme Tz1 on a mutant EGFR gene in eukaryotic cells. The successful identification of catalytic TNAs provides experimental support for TNA as an ancestral genetic material. Additionally, the proof-of-concept demonstration that Tz1 selectively recognizes and catalyzes cleavage reaction of mutant RNA suggests that functional TNAs could be developed into regulatory tools for future biomedical applications.

Chemical structure of TNA and TNA enzyme with RNA cleavage activity

· Screening of TNA enzyme catalyzing 3 '- 5' RNA ligation

Person in charge: Wang Yao

 Threose nucleic acid (TNA) is considered to be a precursor molecule in the process of RNA evolution, because TNA has the capability to form base pairs with RNA, DNA and itself, similar to natural nucleic acids. And the sugar ring of TNA is formed by a simple four-carbon sugar. As the genetic material of the original life form, TNA needs to fold into three-dimensional structure to function as aptamers and nucleases. This project aims to explore whether TNA molecules can catalyze RNA ligation. The identification of TNA ligation with RNA ligase activity, on the one hand, will broaden our understanding of the structure and function of enzyme, and provide experimental evidence for TNA as the precursor of RNA and TNA as the genetic material of the original life form. On the other hand, it can provide RNA editing tools with high biological stability and provide the foundation for the construction of an RNA splicing system based on artificial nucleic acids.

Screening of TNA with RNA ligase activity in vitro

· TNAzyme selection for RNA ligation

Person in charge: Sun Xin

 RNA can be spliced by ribozyme or deoxyribozyme to achieve the purpose of gene editing. The use of natural or artificially RNA lyases usually produce 3’-5'cyclic phosphate ends. When RNA ligation is performed, we usually hope to produce a natural 3'-5' linked structure. Ribozymes or deoxyribozymes are easily degraded by nucleases, which is not conducive to gene editing in vivo. Threonose nucleic acid (TNA) is considered to be a precursor of RNA. It has a different backbone from deoxyribose and is difficult recognized by nucleases. It has high stability and is considered a better gene editing tool. In this topic, we hope to produce TNAzyme that ligates 3’-5' cyclic phosphate ends and 5'hydroxyl through in vitro selection, so as to realize the gene editing of cathepsin A.

Screening of TNA enzyme for RNA ligation in vitro

· TNA-catalysed TNA Ligation

Person in charge: Wang Yueyao

 TNA has been considered as an alternative genetic material of early life forms. TNA is capable of Watson-Crick base pairing with natural DNA, RNA and itself. Previous in vitro selection experiments have demonstrated that TNA could fold into tertiary structures with ligand-binding affinities, which has prompted investigations studying TNA as a potential RNA progenitor or competitor. Among the catalytic activity types of nucleic acid enzymes, the activity that can catalyze self-replication has great advantages in survival and evolution. Therefore, this project aims to explore whether TNA has the ability to catalyze the ligation reaction of TNA. The identification of TNA molecules with TNA ligase activity will provides experimental evidence to support TNA as a potential genetic material of early life forms.

· Preservation of catalytic activity during backbone transition between TNA and RNA

Person in charge: Wei Dongying

 Because of its simple structure, complementary pairing with itself and natural nucleic acids, and folding into a functional secondary structure, TNA is considered as a precursor of RNA by some scholars. However, this hypothesis is still lack of strong evidence, and even if TNA is the precursor of RNA, how to achieve transformation between them, and whether it is gradually replaced or completely replaced remains to be studied. The viewpoint of gradual replacement is more controversial. Due to the structural differences between the two, if TNA nucleotide is partially replaced by ribonucleotide, the original catalytic activity of TNA will be lost. Therefore, this project plans to screen ribozyme and TNAzyme with the same sequence and the same catalytic activity by using Darwin evolution method in vitro to provide the basis for the hypothesis that TNA is the precursor of RNA, and to explore the feasibility of complete substitution hypothesis.

Screening of nucleic acid enzymes compatible with different backbones in vitro

· A semi-synthetic phage that stably carries an unnatural base pair in host cells

Person in charge: Wang Yueyao

 Unnatural base pairs (UBPs) can store and potentially retrieve the increased information, and thereby lay the foundation for achieving the central goal of synthetic biology: the creation of new life forms and functions. This project efforts to introduce UBPs into the genome of phage to construct an artificial phage. This artificial phage can produce novel proteins with site-specifically incorporated noncanonical amino acids (ncAAs) for the prevention and treatment of drug-resistant pathogens. Since the replication of this artificial phage requires the addition of UBPs from an external source, meaning there is less biological risk. In addition, the UBPs with multiple, different functional groups in artificial phage also can help to reveal the molecular mechanism of expansion, expression and assembly of artificial phage in host bacterias.