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Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2′-phosphodiester linkage. Similar results observed for 2′,5′-linked DNA and 2′-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by β-elimination of the 2′-phosphodiester linkage to produce an upstream cleavage product with a 2′-threose sugar and a downstream cleavage product with a 3′ terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications.

 

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3′-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson–Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.

 

Abstract The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises longstanding questions about the order of events in one of life’s most central processes. Here we follow the reaction pathway of a replicative DNA polymerase using time-resolved X-ray crystallography to elucidate the order and transition between intermediates. In contrast to the canonical model, the structural changes observed in the time-lapsed images reveal a catalytic cycle in which translocation precedes catalysis. The translocation step appears to follow a push-pull mechanism where the O-O1 loop of the finger subdomain acts as a pawl to facilitate unidirectional movement along the template with conserved tyrosine residues 714 and 719 functioning as tandem gatekeepers of DNA synthesis. The structures capture the precise order of critical events that may be a general feature of enzymatic catalysis among replicative DNA polymerases. 

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.