For more than 20 years, laboratories around the world have applied the principles of Darwinian evolution to isolate nucleic acid molecules with ligand binding affinity and catalytic activity1-3. DNA and RNA molecules isolated from these selections have been shown to distinguish closely related analogues by a difference of more than 10,000-fold in binding affinity and can accelerate the rate of a chemical reaction by as much as 1010 fold over the uncatalyzed reaction rate4,5. However, despite their ability to fold into shapes with desired functional properties, natural genetic polymers are poor candidates for many diagnostic and therapeutic applications due to their rapid degradation by nucleases6.
This problem can be overcome by removing endogenous DNA- and RNA-degrading enzymes from the sample prior to analysis, but this strategy does not work if the target is a protein or a protein-bound cofactor. Similarly, if the genetic polymers are intended for therapeutic use in vivo, endogenous nucleases will be present in the blood and other biological fluids and tissues that will degrade DNA and RNA before they reach their target. Consequently, numerous chemical modifications have been developed that stabilize the nucleic acid backbone against nuclease digestion7,8. Substitution of the 2′-hydroxyl position of RNA with a methoxy (2′-OMe) or fluoro (2′-F) group, for example, provides resistance against enzymes that utilize the 2′ position to attack the phosphodiester bond. However, care should be taken when modifying oligonucleotides, as chemical changes can adversely affect the functional properties of in vitro selected sequences9.
A more direct approach for advancing functional nucleic acid molecules in the clinic is to develop in vitro selection systems that can be used to evolve synthetic genetic polymers with nuclease-resistant backbones. This approach is desirable, because it avoids the time consuming process of nuclease depletion and sequence re-engineering.
While early work in this area focused on the use of subtle modifications that were tolerated by natural polymerases10,11, new advances in polymerase engineering have made it possible to synthesize unnatural genetic polymers with diverse backbone structures12. These molecules have been termed xeno nucleic acids (or XNA), because they are foreign to biological systems13.
TNA (α-(L)-threofuranosyl-(3′-2′) nucleic acid) is a synthetic genetic polymer in which the natural three-carbon ribose sugar found in RNA is replaced with an unnatural fourcarbon tetrofuranose α-(L) threose sugar. TNA polymers have phosphodiester linkages that occur between the 3′ and 2′ carbon positions, which leads to a backbone repeat unit that is one atom shorter than the backbone unit found in DNA and RNA. However, despite this difference, TNA is able to form stable anti-parallel Watson-Crick duplex structures with complementary strands of DNA RNA, and TNA14,15. The NMR structure of a self-complementary TNA duplex reveals a helical geometry that is similar to A-form RNA, which explains the ability for TNA to cross-pair with DNA and RNA16. The crystal structure of a TNA modified strand indicates that threose prefers a C4′-exo conformation with a rigid backbone and a quasi trans-diaxial orientation of the 3′ and 2′ substituents that allows for DNA and RNA crosspairing by maximizing the spacing between adjacent nucleotides17,18.
Using in vitro selection, we have previously isolated a TNA aptamer that can bind to human thrombin with high affinity and specificity19. Similar results were also obtained for hexose nucleic acid (HNA), in which HNA aptamers were evolved to bind the HIV trans5 activating response RNA element and the protein hen egg lysozyme20. While these aptamers represent the first examples of functional XNA molecules isolated by in vitro selection, growing interest in the field of synthetic genetics suggests that many different types of XNA molecules will be developed in the near future12. As XNA technology continues to advance, it has become important to assess the chemical and biological stability of XNA polymers in environments where these molecules are expected to function.
In their original study, Eschenmoser and colleagues demonstrated that TNA is stable for 8 days at pH 814. To better understand the constraints of TNA polymers, we evaluated the chemical and biological stability of TNA and mixed-backbone (mosaic) TNA-DNA oligonucleotides under a variety of conditions and sequence contexts22.
Accordingly, a need exists for stable, nuclease-resistant oligonucleotides for use in diagnostic and therapeutic applications.