Error-Prone DNA Synthesis on Click-Ligated Templates.

Click ligation is a technology of joining DNA fragments based on azide-alkyne cycloaddition. In the most common variant, click ligation introduces a 4-methyl-1,2,3-triazole (trz) group instead of the phosphodiester bond between two adjacent nucleosides. While this linkage is believed to be biocompatible, little is known about the possibility of its recognition by DNA repair systems or its potential for DNA polymerase stalling and miscoding. Here we report that trz linkage is resistant to several human and bacterial endonucleases involved in DNA repair. At the same time, it strongly blocks some DNA polymerases (Pfu, DNA polymerase ß) while allowing bypass by others (phage RB69 polymerase, Klenow fragment). All polymerases, except for DNA polymerase ß, showed high frequency of misinsertion at the trz site, incorporating dAMP instead of the next complementary nucleotide. Thus, click ligation can be expected to produce a large amount of errors if used in custom gene synthesis.

Aberrant Repair of 8-Oxoguanine in Short DNA Bulges.

The presence of DNA damage can increase the likelihood of DNA replication errors and promote mutations. In particular, pauses of DNA polymerase at the site of damage can lead to polymerase slippage and the formation of 1-2-nucleotide bulges. Repair of such structures using an undamaged DNA template leads to small deletions. One of the most abundant oxidative DNA lesions, 8-oxoguanine (oxoG), was shown to induce small deletions, but the mechanism of this phenomenon is currently unknown. We studied the aberrant repair of oxoG located in one- and two-nucleotide bulges by the Escherichia coli and human base excision repair systems. Our results indicate that the repair in such substrates can serve as a mechanism for fixing small deletions in bacteria but not in humans.

[Calculation of Energy for RNA/RNA and DNA/RNA Duplex Formation by Molecular Dynamics Simulation].

The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the NA-based constructs. Modern advances in computer modeling methods provide the feasibility of these calculations. We have analyzed the possibility of calculating the energy of DNA/RNA and RNA/RNA duplex formation using representative sets of complexes (65 and 75 complexes, respectively). We used the classical molecular dynamics (MD) method, the MMPBSA or MMGBSA approaches to calculate the enthalpy (ΔH°) component, and the quasi-harmonic approximation (Q-Harm) or the normal mode analysis (NMA) methods to calculate the entropy (ΔS°) contribution to the Gibbs energy (ΔG°37) of the NA complex formation. We have found that the MMGBSA method in the analysis of the MD trajectory of only the NA duplex and the empirical linear approximation allow calculation of the enthalpy of formation of the DNA, RNA, and hybrid duplexes of various lengths and GC content with an accuracy of 8.6%. Within each type of complex, the combination of rather efficient MMGBSA and Q-Harm approaches being applied to the trajectory of only the bimolecular complex makes it possible to calculate the ΔG°37 of the duplex formation with an error value of 10%. The high accuracy of predictive calculation for different types of natural complexes (DNA/RNA, DNA/RNA, and RNA/RNA) indicates the possibility of extending the considered approach to analogs and derivatives of nucleic acids, which gives a fundamental opportunity in the future to perform rational design of new types of NA-targeted sequence-specific compounds.

Application of the weighted histogram method for calculating the thermodynamic parameters of the formation of oligodeoxyribonucleotide duplexes.

To date, many derivatives and analogs of nucleic acids (NAs) have been developed. Some of them have found uses in scientific research and biomedical applications. Their effective use is based on the data about their properties. Some of the most important physicochemical properties of oligonucleotides are thermodynamic parameters of the formation of their duplexes with DNA and RNA. These parameters can be calculated only for a few NA derivatives: locked NAs, bridged oligonucleotides, and peptide NAs. Existing predictive approaches are based on an analysis of experimental data and the consequent construction of predictive models. The ongoing pilot studies aimed at devising methods for predicting the properties of NAs by computational modeling techniques are based only on knowledge about the structure of oligonucleotides. In this work, we studied the applicability of the weighted histogram analysis method (WHAM) in combination with umbrella sampling to the calculation of thermodynamic parameters of DNA duplex formation (changes in enthalpy ΔH°, entropy ΔS°, and Gibbs free energy ΔG° 37). A procedure was designed involving WHAM for calculating the hybridization properties of oligodeoxyribonucleotides. Optimal parameters for modeling and calculation of thermodynamic parameters were determined. The feasibility of calculation of ΔH°, ΔS°, and ΔG° 37 was demonstrated using a representative sample of 21 oligonucleotides 4-16 nucleotides long with a GC content of 14-100 %. Error of the calculation of the thermodynamic parameters was 11.4, 12.9, and 11.8 % for ΔH°, ΔS°, and ΔG° 37, respectively, and the melting temperature was predicted with an average error of 5.5 °C. Such high accuracy of computations is comparable with the accuracy of the experimental approach and of other methods for calculating the energy of NA duplex formation. In this paper, the use of WHAM for computation of the energy of DNA duplex formation was systematically investigated for the first time. Our results show that a reliable calculation of the hybridization parameters of new NA derivatives is possible, including derivatives not yet synthesized. This work opens up new horizons for a rational design of constructs based on NAs for solving problems in biomedicine and biotechnology.

A QCM-based rupture event scanning technique as a simple and reliable approach to study the kinetics of DNA duplex dissociation.

Rupture Event Scanning (REVS) is applied for the first time within an approach based on dynamic force spectroscopy. Using model DNA duplexes containing 20 pairs of oligonucleotides including those containing single mismatches, we demonstrated the possibility of reliable determination of the kinetic parameters of dissociation of biomolecular complexes: barrier positions, the rate constants of dissociation, and the lifetime of complexes. Within this approach, mechanical dissociation of DNA duplexes occurs according to a mechanism similar to unzipping. It is shown that this process takes place by overcoming a single energy barrier. In the case where a mismatch is located at the farthest duplex end from the QCM surface, a substantial decrease in the position of the barrier between the bound and unbound states is observed. We suppose that this is due to the formation of an initiation complex containing 3-4 pairs of bases, and this is sufficient for starting duplex unzipping.