Glucose-nucleobase pairs within DNA: impact of hydrophobicity, alternative linking unit and DNA polymerase nucleotide insertion studies† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04850e

Glucose-nucleobase pairs were designed, synthesized and incorporated into duplex DNA. Their stability, structure and polymerase replication was investigated.

: 1 H-NMR assignments of helix glc(Me)-T. 42 - Supplementary references 70 product was purified by silica gel column chromathography using a mixture of hexane, ethyl acetate and triethylamine.

Representative purification HPLC chromatograms of DNA-GNA chimeric oligonucleotides
In bold letters, A and T GNA derivatives and 6dGlc:

MALDI-TOF data of oligonucleotide DNA-GNA chimeric strands
In bold letters, A and T GNA derivatives and 6dGlc: Thermal denaturation methods.
UV-melting curves were measured on a Perkin-Elmer Lambda 750 UV/Vis spectrophotometer.
Absorbance of duplexes in a 1:1 stoichiometric ratio were monitored at 260 nm and the heating rate was set to 1.0 ºC·min -1 from 10 to 80 ºC. The extinction coefficients of the natural oligonucleotide parts, ε nat , was calculated using a oligo calculator (www.ambion.com). The total absorption coefficient was then calculated by simple addition: ε = ε nat1 + ε nat2 . The first derivative of the melting curves was obtained using Origin 8.0 software. To avoid air water condensation samples were measured in a nitrogen atmosphere.
The absorbance versus temperature curves of duplexes were measured at 3. were recorded at 5 °C to reduce the exchange with water. The spectral analysis program Sparky was used for semiautomatic assignment of the NOESY cross-peaks and quantitative evaluation of the NOE intensities. Distance constraints with their corresponding error bounds were incorporated into the AMBER potential energy by defining a flat-well potential term.

Structure Calculations.
Structures were calculated with the SANDER module of the molecular dynamics package AMBER.
Starting models of the conjugate duplexes were built using the program SYBYL. The DNA moieties in the starting models were set to a standard B-canonical structure. These structures were taken as starting points for the AMBER refinement, which started with an annealing protocol in vacuo (using hexahydrated Na + counterions placed near the phosphates to neutralize the system). The resulting structures from in vacuo calculations were placed in the center of a water-box with around 4000 water molecules and 22 sodium counterions to obtain electroneutral systems. The structures were then refined including explicit solvent, periodic boundary conditions and the Particle-Mesh-Ewald method to evaluate long-range electrostatic interactions. Force field parameters for the carbohydrate moieties were taken from GLYCAM. The TIP3PBOX model was used to describe water molecules. The protocol for the constrained molecular dynamics refinement in solution consisted of an equilibration period of 160 ps using a standard equilibration process, followed by four independent 500 ps runs. initio results for weakly interacting systems (that is hydrogen bonding or stacking interactions). [5] Solvent effects in aqueous solution are described with the COnductor-like Screening MOdel (COSMO), which takes effectively into account solute-solvent interactions, cavitation, internal energy and entropy effects of the solvent and yields an estimate of the Gibbs free energies. [6] DNA polymerase primer insertion and extension reactions a. Materials and methods Positions of the oligodeoxynucleotides were located by high efficiency storage phosphor screens and scanned into a Perkin-Elmer Cyclone Plus Phosphorimager.
Steady-state kinetics. Steady-state kinetics for standing-start single nucleotide insertions were carried out as described. [7] The conditions used were the same as for