Enzymatic Synthesis of 7′,5′‐Bicyclo‐DNA Oligonucleotides

Abstract

The selection of artificial genetic polymers with tailor-made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo-DNA triphosphate (i.e., 7',5'-bc-TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7',5'-bc-TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA-dependent DNA synthesis and potentially also XNA-dependent XNA synthesis. However, DNA-dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7',5'-bc-TTP.

Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates

“…Alternatively, “unlocked” nucleic acids (UNAs) synthesized by Therminator DNA Polymerase offer additional substrate functionalization (Dubois et al, 2012). Other examples of functionalized DNAs made by Therminator DNA Polymerase using modified nucleotides include 2′-fluoro-NTPs (Kasuya et al, 2014), glyceronucleotides (gNTPs) (Chen et al, 2009), 7′,5′-Bicyclo-NTPs (Diafa et al, 2017), 3-phosphono-L-Ala-dNMPs (Yang and Herdewijn, 2011; Giraut et al, 2012), 3′-2′-phosphonomethyl-threosyl-NTPs (Renders et al, 2007, 2008), 5′-3′-phosphonomethyl-dNTPs (Renders et al, 2007, 2008), 2′-deoxy-2′-isonucleoside (iNTPs) (Ogino et al, 2010), 3′-deoxyapionucleotide 3′-triphosphates (apioNTPs) (Kataoka et al, 2008, 2011), 5-trifluoromethyl-dUTP (Holzberger and Marx, 2009) and 4′-C-aminomethyl-2′-O-methyl-TTP (Nawale et al, 2012). Kinetic studies of gNTP incorporation by Therminator DNA Polymerase demonstrate that while the rate of gNTP incorporation ( k cat = 0.8–4.7 s −1 ) is similar to dNTPs ( k cat = 2–4.2 s −1 ), gNTPs are bound with >350-fold lower affinity.…”

Recent progress in non-native nucleic acid modifications

“…235 In addition to this steric gate discrimination, the action of polymerases generally requires interaction with the 3′-OH for efficient triphosphate orientation making modifications at this position less easy to incorporate by natural polymerases. Despite this, polymerases have been found to incorporate a wide range of sugar modifications such as 2′-fluoroarabinonucleic acids (FANA), 224 LNA, 236 7,5-bicylo-DNA, 237 and (3′-2′) α-L-threose nucleic acid (TNA), 238, 239 among others. Backbone modifications of the phosphodiester linker involving replacement of the nonbinding negatively charged oxygen with a negatively charged species such as PS and borane (BH 3 -) have been successfully achieved enzymatically, albeit perhaps leading to some copy error issues with PS.…”

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate

“…S8b). We next conducted bypass experiments using the more permissive polymerase Therminator [59][60]61 which is known to recognize UBPs. 62 Under these conditions, the formation of the n+6 product was observed by gel electrophoresis (Fig.…”