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Abstract Inspired by nature, chemists have spent the last 50 years systematically designing and synthesizing a vast array of sugar-modified nucleic acids, so-called xenonucleic acids (XNAs), collectively forming what we now describe as the XNA alphabet. Within the alphabet, systems can be categorized into two major groups: those capable of interacting with natural nucleic acids and those that do not cross-pair with DNA or RNA. The sugar component of XNAs plays a crucial role in defining their conformational space, which, in turn, influences their hybridization properties and potential applications across biotechnology and synthetic biology. This review provides an overview of sugar-modified XNA systems developed to date as well as the geometric parameters and physicochemical principles that have enhanced our understanding of XNA conformational behavior, particularly in relation to their orthogonality to (i.e. inability to cross-pair with) natural nucleic acids. These insights are essential for developing a more rational approach to key processes such as XNA replication and evolution, ultimately paving the way for applications in areas including synthetic genetics, nucleic acid therapeutics, diagnostics, and nanotechnology. 

Polymerases are among the most powerful tools in the molecular biology toolbox; however, access to large quantities of chemically modified nucleoside triphosphates for diverse applications remains hindered by the need for purification by high-performance liquid chromatography (HPLC). Here, we describe a scalable approach to modified nucleoside triphosphates that proceeds through a P(III)−P(V) mixed anhydride inter-mediate obtained from the coupling of a P(III) nucleoside phosphoramidite and a P(V) pyrene pyrophosphate reagent. The synthetic strategy allows the coupling, oxidation, and deprotection steps to proceed as stepwise transformations in a single one-pot reaction. The fully protected nucleoside triphosphates are purified by silica gel chromatography and converted to their desired compounds on scales exceeding those achievable by conventional strategies. The power of this approach is demonstrated through the synthesis of several natural and modified nucleoside triphosphates using protocols that are efficient and straightforward to perform. 

Abstract 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. 

Abstract 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.