α-L-Threose Nucleic Acids as Biocompatible Antisense Oligonucleotides for Suppressing Gene Expression in Living Cells.

Due to the chemical simplicity of α- L-Threose nucleic acid (TNA) and its ability to exchange genetic information between itself and RNA, it has been raised significant interests in TNA as RNA ancestor. We herein explore the biological properties and evaluate the potency of sequence-designed TNA polymers to suppress gene expression in living environments. We found that sequence-specific TNA macromolecules exhibit strong affinity and specificity towards the complementary RNA targets, are highly biocompatible and non-toxic in living cell system, and readily enter a number of cell lines without using transfecting agents. Particularly, TNA exhibited much stronger enzymatic resistance toward fetal bovine serum or human serum as compared to traditional antisense oligonucleotide which mean that the intrinsic structure of TNA is thoroughly resistant to biological degradation. Importantly, the efficacy of TNA molecule with GFP target sequence (anti-GFP TNAs) as antisense agents was firstly demonstrated in living cells in which these polymers revealed high antisense activity in terms of the degree of inhibition of GFP gene expression. The GFP gene inhibition studies in HeLa and HEK293 cells characterize sequence-controlled TNA as a functional biomaterials and a valuable alternative to traditional antisense oligonucleotide such as PNAs, MOs and LNAs for a wide range of applications in drug discovery and life science research. Additionally, we also firstly reported the cost-efficient approach to synthesize the four TNA phosphoramidite monomers using 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite as a key reagent. Furthermore, by increasing the frequency of the de-blocking and coupling reaction together with extending their reaction time in each synthesis cycle, sequence-controlled TNAs can be easily synthesized in a quantitative yield and high purify.