Development of Threose Nucleic Acid-Based Reagents for Biomedical Applications


Student thesis: Doctoral Thesis

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Award date7 Jan 2022


(3’,2’)-α-L-threose nucleic acid (TNA) is a chemically modified xeno nucleic acid (XNA), in which the five-carbon ribose in DNA and RNA is replaced by the four-carbon threose. Though the backbone repeat unit is one atom shorter than that of DNA and RNA, TNA is capable of forming stable Watson-Crick antiparallel duplex structures with complementary strands of DNA, RNA, and itself. The attractive characteristics of chemical simplicity, high specificity and binding affinity towards DNA and RNA, capability to fold into tertiary structures, and remarkable biological stability make TNA a promising candidate in the development of XNA-based reagents for biological studies and biomedical applications.

Here, we demonstrated that the physiological stable TNA oligonucleotides (ONs) exhibited enhanced cellular uptake in various cell lines and displayed time-, concentration-dependent penetration into the three-dimensional (3D) cell culture model. In addition, we investigated the pharmacokinetics and biodistribution of TNA ONs. After the intravenous administration into Balb/c mice, the TNA ONs exhibited rapid clearance from blood in hours. The ex vivo fluorescence imaging revealed that the TNA ONs dominantly accumulated in the kidneys with a time-dependent decrement, suggesting the excretion of TNA ONs from the body through the renal filtration system. The preliminary in vivo biosafety measurements indicated that the intravenous administration of TNA ONs induced negligible hematological and histopathological abnormality or dysfunction to the kidneys, indicating the excellent biocompatibility of TNA ONs.

Based on the high binding affinity with RNA and excellent biological stability, we designed and synthesized a sequence-defined TNA polymer that is complementary to the certain nucleotide region of the target anti-apoptotic BcL-2 gene. Comparing to the scramble TNA, anti-BcL-2 TNA significantly suppressed target mRNA and protein expression in MCF-7 cancerous cells and showed antitumor activity in carcinoma xenografts, resulting in inhibition of cancer cell proliferation and tumor growth.

In addition, we successfully designed and fabricated TNA probes based on strand displacement reactions for sensitive microRNA (miRNA) detection and intracellular miRNA imaging. The developed TNA probes indicated high specificity and selectivity towards target miRNAs and could differentiate one to two base mismatches in the molecules. Compared to DNA probes, these TNA probes exhibited superior nuclease stability, thermal stability, and exceptional storage ability for long-term cellular studies. TNA probes could not only be efficiently taken up by cells with insignificant cytotoxicity for dynamic detection of target miRNAs, but also differentiate the distinct target miRNA expression levels in different cell lines.