Search for: All records

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.