Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases.

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

Enzymatic Synthesis of Backbone-Modified Oligonucleotides Using T4 DNA Ligase.

T4 DNA ligase in high concentrations of certain crowding agents and cosolutes catalyzes the synthesis of a series of backbone-modified oligonucleotides that are difficult to obtain chemically. Backbone-modified nucleic acids are often enzymatically and chemically more stable, making them interesting as potential diagnostic or therapeutic agents, as a biosafety tool, or in nanotechnology. In this article, we describe a small-scale experiment to probe the efficiency of the ligation reaction of modified oligonucleotides in the presence of 3 M betaine and 10% PEG 8000, followed by large-scale ligation with subsequent isolation of the ligated oligonucleotide. The correct product formation can be verified using denaturing polyacrylamide gel electrophoresis and mass spectrometry. © 2019 by John Wiley & Sons, Inc.

XNA ligation using T4 DNA ligase in crowding conditions.

T4 DNA ligase is capable of ligating 2'OMe-RNA duplexes, HNA, LNA and FANA mixed sequences in the presence of 10% w/v PEG8000 and 3 M betaine. The enzymatic joining of oligonucleotides containing multiple consecutive XNA nucleotides at the ligation site has not been reported before.

Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence.

The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 2050(1). Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non-growing antibiotic-tolerant 'persister' state(2-6). Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment(7). Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20-100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models(8-10), we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of 'bet-hedging' theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.