A threose nucleic acid (TNA) enzyme catalyzing native 3′-5′ ligation of RNA

Person in charge: Wang Juan

Source link: https://doi.org/10.1021/jacs.5c07235

Threose nucleic acid (TNA) is a synthetic genetic polymer of both prebiotic significance and practical utility. Identification of TNA molecules with enzymatic activities (TNAzymes) not only lends experimental support for TNA as a potential primitive catalyst but also offers intrinsically stable biotechnological and biomedical molecular tools. Here, we report the in vitro selection of TNAzymes capable of catalyzing the native 3′-5′ ligation of two RNA oligonucleotides. The Zn²⁺-dependent TNAzyme facilitates the formation of a canonical phosphoester bond between a terminal 3′-hydroxyl group on one substrate and a 5′-triphosphate on the other. Under optimal conditions (pH 7.3 and 23 °C), the TNAzyme exhibits a catalytic rate constant of 0.39 h^-1. Lastly, we demonstrate that the TNAzyme-catalyzed ligation of two RNA fragments could yield a functional RNA product such as a ribozyme. These findings showcase the potential role of TNA as a primordial catalyst during the emergence of the RNA world, as well as its prospective application in RNA synthesis.

TNAzyme with capacity to promote bond formation between 3′-hydroxyl and 5′-triphosphate groups

Selection of RNA-cleaving TNA enzymes for cellular Mg²⁺ imaging

Person in charge: Gao Mingmei, Wei Dongying

Source link: https://doi.org/10.1002/cbic.202200651

Catalytic DNA-based fluorescent sensors have enabled cellular imaging of metal ions such as Mg²⁺. However, natural DNA is prone to nuclease-mediated degradation. Here, we report the in vitro selection of threose nucleic acid enzymes (TNAzymes) with RNA endonuclease activities. One such TNAzyme, T17-22, catalyzes a site-specific RNA cleavage reaction with a k_cat of 0.017 min^-1 and K_M of 675 nM. A fluorescent sensor based on T17-22 responds to an increasing concentration of Mg²⁺ with a limit of detection at 0.35 mM. This TNAzyme-based sensor also allows cellular imaging of Mg²⁺. This work presents the first proof-of-concept demonstration of using a TNA catalyst in cellular metal ion imaging.

TNAzyme-based fluorescent sensorenables Mg²⁺ imaging in living cells

A nucleic acid sequence that is catalytically active in both RNA and TNA backbones

Person in charge: Wei Dongying

Source link: https://doi.org/10.1021/acssynbio.2c00479

Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle in vitro selection to identify nucleic acid catalyst sequences that are active in both TNA and RNA backbones. One such nucleic acid enzyme with exchangeable backbone (CAMELEON) catalyzes an RNA cleavage reaction when prepared as TNA (T) and RNA (R). Further biochemical characterization reveals that CAMELEON R and T exhibit different catalytic behaviors such as rate enhancement and magnesium dependence. Structural probing and mutagenesis experiments suggest that they likely fold into distinct tertiary structures. This work demonstrates that the catalytic activity can be preserved during backbone transition from TNA to RNA and provides further experimental support for TNA as an RNA precursor in evolution.

Maintenance of catalytic activity between RNA and TNA

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

Person in charge: Wang Yueyao, Wang Yao

Source link: https://doi.org/10.1038/s41557-021-00847-3

Threose nucleic acid has been considered a potential evolutionary progenitor of RNA because of its chemical simplicity, base pairing properties and capacity for higher-order functions such as folding and specific ligand binding. Here we report the in vitro selection of RNA-cleaving threose nucleic acid enzymes. One such enzyme, Tz1, catalyses a site-specific RNA-cleavage reaction with an observed pseudo first-order rate constant (k_obs) of 0.016 min^-1. The catalytic activity of Tz1 is maximal at 8 mM Mg²⁺ and remains relatively constant from pH 5.3 to 9.0. Tz1 preferentially cleaves a mutant epidermal growth factor receptor RNA substrate with a single point substitution, while leaving the wild-type intact. We demonstrate that Tz1 mediates selective gene silencing of the mutant epidermal growth factor receptor in eukaryotic cells. The identification of catalytic threose nucleic acids provides further experimental support for threose nucleic acid as an ancestral genetic and functional material. The demonstration of Tz1 mediating selective knockdown of intracellular RNA suggests that functional threose nucleic acids could be developed for future biomedical applications.

Tz1 with RNA endonuclease activity

A Threose Nucleic Acid Enzyme with RNA Ligase Activity

Person in charge: Wang Yao

Source link: https://doi.org/10.1021/jacs.1c02895

Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2′–5′ phosphoester bond between a 2′,3′-diol and a 5′-triphosphate group, with a k_obs of 1.1 × 10^-2 min^-1 (40 mM Mg²⁺, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2′–5′ linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.

Catalytic TNA molecule with RNA ligase activity

Selection of threose nucleic acid aptamers to block PD-1/PD-L1 interaction for cancer immunotherapy

Person in charge: Li Xintong

Source link: https://doi.org/10.1039/d0cc06032a

Threose nucleic acid (TNA) aptamers were selected in vitro to bind PD-L1 protein and inhibit its interaction with PD-1. These biologically stable TNA aptamers bound target proteins with nanomolar affinities, and effectively blocked PD-1/PD-L1 interaction in vitro. After injection into a colon cancer xenograft mouse model, the TNA aptamer N5 was specifically accumulated at the tumour site, and significantly inhibited tumour growth in vivo.

In vitro selected TNA aptamer inhibits PD-1/PD-L1 interaction