Polymerase synthesis of four-base DNA from two stable dimeric nucleotides

Abstract

We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.

Recent progress in non-native nucleic acid modifications

“…These dimeric pentaphosphate analogues are also readily tolerated by DNA polymerases such as the Klenow fragment of DNA Polymerase I (exo -) and therefore represent valid alternatives to dN*TPs. 249 Instead of increasing and modifying the polyphosphate moiety, multiple nucleosides can be integrated in triphosphate analogues. Such trinucleotide triphosphates (triplets) can readily be polymerised by a ribozyme obtained by in vitro evolution methods (Fig.…”

Beyond Triphosphates: Reagents and Methods for Chemical Oligophosphorylation

“…However, this methodology produces a distribution of nucleoside oligophosphates, hampering its utility. The first electrophilic activation of TriMP comes from Taylor, who in situ activated TriMP with mesitylenesulfonyl chloride and nucleophilic tertiary amines, ,,, a reaction which has also been utilized by Kool . TriMP has been activated similarly with (7-azabenzotriazol-1-yloxy)­tripyrrolidinophosphonium hexafluorophosphate, (PyAOP), and in this case the resulting activated TriMP derivative could be structurally characterized .…”

Genetic Diversity for Accelerating Microbial Adaptive Laboratory Evolution

“…DNA polymerases play an important role when modified nucleoside triphosphates (dNTPs) are used to extend a DNA primer hybridized to a complementary DNA template . There are many types of DNA polymerases in different microorganisms, which all have different enzymological characteristics and intrinsic mutation rates.…”

“…DNA polymerases play an important role when modified nucleoside triphosphates (dNTPs) are used to extend a DNA primer hybridized to a complementary DNA template. 47 There are many types of DNA polymerases in different microorganisms, which all have different enzymological characteristics and intrinsic mutation rates. For example, E. coli has five DNA polymerases, among which DNA polymerase III is responsible for genome replication, encompassing both the polymerization and errorproofing functions.…”