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

Abstract

Because of the chemical simplicity of α-l-threose nucleic acid (TNA) and its ability to exchange genetic information between itself and RNA, it has attracted significant interest as the RNA ancestor. We herein explore the biological properties and evaluate the potency of sequence-designed TNA polymers to suppress the gene expression in living environments. We found that sequence-specific TNA macromolecules exhibit strong affinity and specificity toward the complementary RNA targets, are highly biocompatible and nontoxic in a 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 oligonucleotides, which means that the intrinsic structure of TNA is thoroughly resistant to biological degradation. Importantly, the efficacy of the TNA molecule with green fluorescent protein (GFP) target sequence (anti-GFP TNAs) as antisense agents was first 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 biomaterial and a valuable alternative to traditional antisense oligonucleotides such as peptide nucleic acids, phosphorodiamidate morpholino oligomers, and locked nucleic acids for a wide range of applications in drug discovery and life science research. Additionally, we also first 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 deblocking and coupling reactions together with extending their reaction time in each synthesis cycle, sequence-controlled TNAs can be easily synthesized in a quantitative yield and high purity.

Progress toward Understanding the Interactions between DNA Nanostructures and the Cell

“…A decade ago, chemists in the field of nanomedicine pioneered oligonucleotides with various chemical modifications to enhance their stability against nucleases, such as peptide nucleic acids, locked nucleic acids, and PS backbone. For instance, Liu et al reported the synthesis of α‐ l ‐threose nucleic acid (TNA) polymers with higher resistance against serum proteins than conventional PO‐based oligonucleotides in 2018 . These TNA polymers show strong binding affinity to complementary RNA targets and can inhibit the expression of a target gene.…”

Nucleic Acid–Based Therapeutics in Orphan Neurological Disorders: Recent Developments

“…Synthetic polymers of α-l-threose–based nucleic acids (TNA; Figure 1 ), containing a 4-carbon sugar as opposed to 5-carbon ribose found in natural nucleic acids, show strong binding affinity toward the complementary target RNAs, high nuclease resistance, and low toxicity; have easy and cost-efficient synthesis; and can be taken up by cells without transfection agents ( Liu et al, 2018 ). TNA-based aptamers and ASOs with biological activity have been introduced.…”

Activation of Innate Immune Responses by a CpG Oligonucleotide Sequence Composed Entirely of Threose Nucleic Acid

“…Recent advances in TNA synthesis using engineered polymerases have allowed for the selection of TNAs that target human thrombin [16] and HIV reverse transcriptase, opening the door for selections against a variety of other protein targets. In addition, a recent study demonstrated that antisense TNA oligonucelotides are able to effectively suppress GFP expression in living cells [17].…”

“…Due to a high level of resistance to serum nucleases [15] and recent advances in the use of engineered polymerases that can copy genetic information back and forth between TNA and DNA [1,16], TNA has become an attractive tool for a variety of applications. In fact, in vitro selection of TNA aptamers targeting human thrombin [16], HIV reverse transcriptase [5], and ochratoxin A [6] has recently been reported, as well as an antisense TNA targeted to green fluorescent protein (GFP) that suppresses GFP expression in live cells [17].…”